Plant gene specifying acetyl coenzyme A carboxylase and transformed plants containing same

ABSTRACT

DNA sequence of an acetyl Coenzyme A carboxylase from plants are inserted into the genome of plants in sense or antisense orientation in order to inhibit expression of the gene product of the endogenous ACCase gene, resulting in reduced conversion of the enzyme&#39;s substrate, acetyl Coenzyme A, to fatty acid synthesis, leaving the substrate available for diversion into other biosynthesis pathways. One such diversion may be accomplished by providing the plant genome with genes specifying the synthesis of polyhydroxyalkanoate polymers.

This invention relates to a plant gene specifying the enzyme acetylCoenzyme A carboxylase (ACCase) and to plant genomes geneticallytransformed with the said gene. Particularly, but not exclusively, theinvention relates to ACCase genes from plants of the Brassica species,especially Brassica napus (oilseed rape) and control of expression ofthe gene by Brassica plants which are genetically transformed with thegene or its antisense configuration.

Acetyl Coenzyme A carboxylase is one of the genes involved in thesynthesis of oil by oil-producing crops such as oilseed rape. Variationof the expression of that gene leads to alteration in the quantityand/or quality of the oil produced.

An object of the invention is to provide a gene specifying ACCase inplants.

According to the present invention there are provided partial cDNAsspecifying ACCase, isolated from seed of Brassica napus, having thenucleotide sequences set forth in FIGS. 6 and 12, and variations thereofpermitted by the degeneracy of the genetic code.

The invention further provides the partial cDNA, isolated from wheatgerm, having the nucleotide sequence set forth in FIG. 4, and variantsthereof permitted by the degeneracy of the genetic code.

Also provided by this invention is the full length genomic DNAspecifying ACCase from Arabidopsis thaliana having the nucleotidesequence set forth in FIG. 8, and variants thereof permitted by thedegeneracy of the genetic code.

The invention further provides the following clones, inserted inEscherichia coli, strain DHα hosts, which have been deposited with theNational Collection of Industrial & Marine Bacteria, 23 St. Machar Road,Aberdeen, AB2 1RY, United Kingdom, on Mar. 25, 1993, under theprovisions of the Budapest Treaty on the Deposit of Microorganisms forPatent Purposes, details of which are as follows:

1. Plasmid pK111, Accession No. NCIB 40553

2. Plasmid pKLU81, Accession No.NCIB 40554

3. Plasmid pRS1, Accession No. NCIB 40555

The present invention also provides genetically transformed plants,plant cells and plant parts, containing a DNA of the invention orfragment thereof in sense orientation or a complete or partial sense orantisense variant thereof.

It is preferred that the plant be of a species which producessubstantial quantities of oil, rather than starch. Such plant speciesare well known and are simply referred to as “oil-seed” crops andinclude, oilseed rape, canola, soya and sunflower. Methods for thegenetic transformation of many oil crops are known; for example,transformation by Agrobacterium tumefaciens methods are suitable formost. Such methods are well-described in the literature and well-knownand extensively practised in the art.

In our International Patent Application Number WO 92/19747, published onNov. 12, 1992, we describe the biosynthesis of polyhydroxybutyrate fromthe substrate, acetyl-CoA. This activity involves three enzyme-catalysedsteps. The three enzymes involved are β-ketothiolase, NADP linkedacetoacetyl-CoA reductase, and polyhydroxybutyrate synthase, the genesfor which have been cloned from Alcaligenes eutrophus (Schubert et al,1988, J Bacteriol, 170). In our international application we describethe cloning of these three gene into oil-synthesising plants.

However, the synthesis of fatty acids which are the building blocks ofplant oils utilise the substrate acetyl Coenzyme A which is the samesubstrate required by the polyhydroxyalkanoate genes. By virtue of thepresent invention we provide means for down-regulating the fatty acidsynthesis by inhibiting ACCase thereby leaving the acetyl CoA availablefor conversion to polyhydroxyalkanoates.

Methods for the regulation of gene expression are well-known in the art.Two principal methods are commonly employed, these being referred toloosely as “sense” and “antisense” regulation. In antisense regulation agene construct is assembled which, when inserted into a plant cell,results in expression of a messenger RNA which is of complementarysequence to the messenger produced by a target gene. The theory is thatthe complementary RNA sequences form a duplex thereby inhibitingtranslation to protein. The complementary sequence may be equivalent inlength to the whole sequence of the target gene but a fragment isusually sufficient and is more convenient to handle. In sense regulationa copy of the target gene is inserted into the plant genome. Again thismay be a full length or partial sequence. A range of phenotypes isobtained from which individuals in which the expression of the proteinencoded by the target gene is inhibited may be identified and isolatedas may individuals where expression of the gene product is increased.Sense regulation using partial sequences tends to favour inhibition. Themechanism is not well understood. Reference is made to European PatentApplication No. 140,308 and U.S. Pat. No. 5,107,065 which are bothconcerned with antisense regulation and International Patent ApplicationNo. WO 90/12084 which describes sense regulation. The invention permitsthe following genetic modifications to be effected:

1. The clones of the invention may be used to probe plant DNA (genomicor cDNA libraries) to obtain homologous sequences. These may betruncated or full length cDNAs or genomic DNAs for ACCase genes from,for example, wheat, or oil crops such as rape, canola, soya, sunflower,maize, oil palm and coconut.

2. Partial cDNAs of rape seed ACCase may be used in conjunction with aplant-recognised promoter to create an expression cassette (partialsense or antisense) for use in transforming rape plants to down-regulateproduction of the ACCase enzyme. This will give plants with a lower oilcontent or oil of altered quality. The same cassette can be used todown-regulate the production of ACCase enzyme in other plants of theBrassica species. cDNAs isolated from other crops can be used to createexpression cassettes (partial, sense or antisense) for use intransformation of these crops in order to modify the oil content.

Down-regulation of oil synthesis (in rape or other oil crops) can beused to divert the substrate, acetyl Coenzyme A, into synthesis ofalternative storage materials such as starch, protein, or novel polymersintroduced by genetic modification, for example polyhydroxyalkanoates.

3. Full length clones of rape or Arabidopsis ACCase DNA can be used tocreate expression cassettes, either with powerful promoters, or byinserting extra gene copies, to promote over-expression of ACCase inrape or other oil crops, leading to plants with enhanced oil content inthe seed. The ACCase DNA may also be put under the control of aseed-specific promoter such as the napin promoter, which has a differentwindow of expression from the ACCase promoter during seed development.In this way the period over which ACCase is expressed in the developingseed is extended, and the oil content of the seeds increased.

4. Genomic DNAs of rape ACCase can be used to recover the promoter ofthe ACCase gene. This promoter can be used to generate RNA in atissue-specific and developmentally regulated fashion. The promoter sogenerated may promote the expression of ACCase, or it may control theexpression of a gene construct placed after it (for example thestructural gene of a different enzyme) which will then be expressedspecifically in the developing seed.

5. The full length cDNA and genomic DNA of rape or Arabidopsis ACCasecontain a sequence between the translation start site and the N-terminalsequence of the mature protein, known as a “transit peptide” sequence.This directs the gene product to the plastids and is cleaved off duringimport of the protein into the plastids. This transit peptide sequencemay be used in gene fusions to direct different gene products to theplastids.

6. Monocotyledonous plants, such as wheat, barley, maize and rice, arenormally sensitive to the aryloxyphenoxy-propionate and alkylketoneherbicides to which the dicotyledonous plants are normally resistant.Monocots with resistance to these herbicides may be created by:

(a) transforming ACCase from a dicotyledonous species such as rape andArabidopsis, into the monocot genome;

(b) overexpression of the ACCase in a monocot; or,

(c) mutagenesis of ACCase and insertion of the mutant gene into amonocot.

7. It is believed that ACCase activity exists in both the plastid andthe cytosol. Partial cDNAs of rape seed ACCase of this invention may beused in conjunctipon with a plant-recognised promoter to create anexpression cassette (partial sense or antisense) for use in transformingplants to down-regulate production of the cytosolic ACCase. This willalter oil quality by inhibiting production of long chain fatty acids)chain length greater than about C18).

8. A second plastid form of ACCase has been identified in plants. ThisACCase is composed of dissociable sub-units for transcarboxylase, biotincarrier protein (BCP) and biotin carboxylase (BC). The transcarboxylasegene is encoded by the chloroplast genome; BCP and BC are nuclearencoded. Sequence homology between the cDNAs of the invention and theBCP and BC may be used to isolate BCP and BC. Sense and antisenseconstructs may be raised against BCP and BC in order to effectdown-regulation of these genes.

9. The cDNAs of the invention may themselves have sufficient homologywith the BCP and BC genes to be used directly for the down-regulation ofthese genes.

We have prepared a poly dT primed cDNA library from developing rape seedand have obtained another from developing wheat embryo. These librarieshave been probed with DNA fragments isolated earlier from a partiallength maize leaf ACCase DNA (pA3) and partial length cDNA clonesspecifying rape seed ACCase (pRS1) and wheat germ ACCase (pK111) havethereby been selected and sequenced.

A DNA fragment isolated from the partial length rape ACCase DNA was thenused to probe a genomic DNA library prepared from Arabidopsis thalianaand a full length Arabidopsis genomic DNA selected and sequenced.

The sequence of the Arabidopsis genomic DNA was used to generatespecific probes by PCR. These were used to screen a random primed cDNAlibrary from rape seed and two further rape ACCase partial cDNAs werethus isolated.

The full length Arabidopsis ACCase genomic DNA may then be used to probea genomic library from rape and the full length rape ACCase genomic DNAselected and sequenced.

That the clones were indeed of ACCase genes was confirmed as follows:

The deduced amino acid sequence for wheat ACCase cDNA shows completehomology in four regions of sequence to the amino acid sequencesobtained from four peptides isolated from the ACCase enzyme purifiedfrom wheat embryo. The deduced amino acid sequence shows high homologywith both the rat and chicken ACCase genes. High homology at the aminoacid level with maize leaf ACCase was found, with two sections of 48amino acids completely conserved.

The deduced amino acid sequence from the rape seed partial cDNA (pRS1)sequence shows high homology to the sequences of the maize leaf cDNA andthe chicken, rat, yeast and algal ACCase genes.

The deduced amino acid sequence from the Arabidopsis genomic DNA showshigh homology with the rat, chicken and yeats ACCase genes. Highhomology with the amino acid sequence of the rape seed ACCase partialcDNA (pRS1) was found, with one section of 48 amino acids almostcompletely conserved.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described with reference to the accompanyingdrawings which show:

FIG. 1 shows the elution profiles of wheat embryo ACCase fromQ-Sepharose (FIG. 1A) and Blue-Sepharose (FIG. 1B) during purificationof the enzyme. The dotted line represents the sodium chloride gradientconcentration and the activity of ACCase, represented by the boxes, wasmeasured as described hereinbelow;

FIG. 2A shows an SDS PAGE gel of wheat embryo ACCaseshowing thealteration in mobility caused by the binding of streptavidin. Lane 1contains 500 ng myosin (200 kDa); lane 2 contains 10 μl PostBlue-sepharose material without Streptavidin; and, lane 3 contains 10 μlPost Blue-sepharose material with streptavidin. ACCase is indicated byasterisks (*) at its normal migration and that of theACCase/streptavidin complex respectively.

FIG. 2B shows an SDS PAGE gel of purified wheat embryo ACCase, with the220 kd band taken for sequencing indicated. Lane 1 contains 1 μl PostBlue-sepharose material and lane 2 contains 10 μl Post Blue-sepharosematerial;

FIG. 3 shows a comparison of four sections of amino acid sequencededuced from the pK111 wheat ACCase cDNA with the amino acid sequencesobtained from four peptides isolated from the purified wheat embryoACCase enzyme (SEQ ID NOS:1-8;

FIG. 4 shows the sequence of the sense strand of the wheat embryo ACCaseclone pK111 (SEQ ID NO:9), with three-phase translation shown (SEQ IDNO:10). The sequences homologous with the peptide amino acid sequencesare underlined;

FIG. 5 shows a dot matrix plot of the deduced amino acid sequence ofwheat ACCase clone pK111 against that of the maize ACCase clone pA3;

FIG. 6a shows the derived amino acid sequence (SEQ ID NO:11) from therape cDNA encoding the transcarboxylase domain of ACCase. The amino acidsequence is translated from the first open reading frame shownpictorially. The full vertical lines represent stop codons and the halfvertical lines ATG sequences.

FIG. 6b shows the nucleotide sequence (SEQ ID NO:12) of the cDNA clonepRS1, corresponding to the trans carboxylan domain of ACCase.

FIG. 7 shows the rape transcarboxylase domain comparison with knownACCase sequences. The Dot Matrix (DNA Strider, Stringency 9 Window 21)of derived rape ACCase amino acid sequence (transcarboxylase domain) iscompared against rat, yeast and algal (Chlorella) ACCase.

FIG. 8 shows the 5′ sequence (SEQ ID NO:13) from the sense strand of theArabidopsis genomic subclone pKLU81, with three phase translation shown(SEQ ID NO:14).

FIG. 9 shows the 3′ sequence (SEQ ID NO:15) from the sense strand of theArabidopsis genomic subclone pKLU81, with three-phase translation shown(SEQ ID NO:16).

FIG. 10 shows a comparison of the Arabidopsis pKLU81 5′ translated openreading frame (SEQ ID NO:17) with the sequences of rat and chickenACCase genes (SEQ ID NOS:18 -19) obtained from SWISSPROT database.

FIG. 11 shows the assignment of domain order to higher plant ACCase.

FIG. 11A is a schematic diagram showing the yeast ACCase domain ordersrelative to the sequenced regions (hatched boxes) of the Arabidopsisgenomic clone. The areas of sequenced genomic clone are named A-F foreasy identification in the text.

In FIG. 11Bi) the translated open reading frame (SEQ ID NO:20) from areaAii is shown in direct comparison with a region from the biotincarboxylase domain of yeast (SEQ ID NO:21). Boxed regions representamino acid identity.

In FIG. 11Bii) the translated open reading frame (SEQ ID NO:22)corresponding to the biotin binding site in area C is shown in directcomparison with the biotin binding site of yeast (SEQ ID NO:23). Boxedregions represent amino acid identity.

FIG. 11Biii) shows a DNA sequence comparison by dot matrix (DNA Strider,Stringency 15 Window 23) of the rape transcarboxylase domain of ACCaseand areas E/F from the Arabidopsis genomic clone.

FIG. 11C shows the nucleotide sequences (SEQ ID NOS:24-30) of Regions A,Aii, B, C, D, E, F, Arabidopsis genomic clone pKLS2.

FIG. 12 shows the rape ACCase biotin binding domain sequence.

FIG. 12Ai) shows the derived amino acid sequence (SEQ ID NO:31) from therape cDNA encoding the ACCase biotin binding domain. The actual biotinbinding site is shown underlined.

FIG. 12Aii) shows the direct comparison of the biotin binding site withthat of the corresponding sequence of yeast ACCase. The boxed regionsrepresent amino acid sequence identity.

FIG. 12B shows the dot matrix comparison (DNA Strider, Stringency 9Window 21) of derived rape ACCase amino acid sequence (biotin bindingdomain) against yeast ACCase.

FIG. 12C shows the full combined nucleotide sequence (SEQ ID NO:32) ofpRS6 and pRS8.

FIG. 13 shows ACCase Southern blot analyses of rape and Arabidopsisgenomic DNA. Restrriction endonuclease digested DNA was hybridised tothe Arabidopsis ACCase genomic clone by Southern blot. Hybridisation andwashing conditions were carried out as described in materials andmethods. The blot shown was exposed for 5 days, further exposureprovided no extra information. Both λ HindIII and OX 174 HaeIII DNAmarkers (indicated on the left hand side) were run on the same 1% geland viewed by ethidium bromide staining/UV.

FIG. 14 shows Northern blot analysis of rape ACCase. In FIG. 14A thegraph shows the oil content as total fatty acid (mg/seed) in relation tothe stage of rape embryogenesis. Details of the analysis method arepresented in materials and methods. The three Northern blots shown,relating to the different stages of embryogenesis, are all derived fromthe same blot after successive stripping. The probes used were asindicated in the text and the amount of polyA+RNA was 5 μg for eachstage. Hybridisation and washing conditions were as in materials andmethods. Exposure was for 7 days.

In FIG. 14B the probe used in the Northern blot shown was the rapetranscarboxylase domain cDNA derived from an embryo library. 1 μg ofpoly A+RNA from 29 days post anthesis embryos and young leaf was usedfor the blot. Hybridisation and washing conditions were as in materialsand methods. Exposure was for 7 days. Molecular weight markers wereviewed by ethidium bromide/UV.

MATERIALS AND METHODS 1.0 Protein Purification and Amino Acid SequenceData

1.1 Assay for ACCase

Acetyl CoA Carboxylase activity was assayed by incorporation ofradioactivity from ¹⁴C-bicarbonate into non-volatile malonyl CoA(Hellyer et al 1986).

1.2 SDS Poly-Acrylamide Gel Electrophoresis

All SDS PAGE gels consisted of a 3% stacking gel with a 7.5% running gelon a mini Biorad Protean gel kit unless otherwise stated. The buffersystem used was that of Laemmli et al (1970) unless otherwise stated.All gels used in separating peptides for sequencing were pre-run in thepresence of 200 μM thioglycolic acid in the running buffer.

2.0 Cloning for Wheat/Rape/Arabidopsis ACCase

2.1 Preparation of competent XL1-Blue and KW251

Escherichia coli cells XL1-Blue and KW251 cells were grown overnight in50 ml LB media/0.2% Maltose/50 μ/ml Tetracycline/10 mM MgSO₄. The cellswere spun down at 3000 g for 10 mins and the cell pellet taken up in 2.5ml 10 mM MgSO₄ and stored at 4° C. Cells were used fresh for primaryscreening and no older than one week for subsequent screening.

2.2 cDNA Libraries

2.2.1 Wheat

The cDNA library used (gift of Dr Charles Ainsworth, Wye College,London) was generated using the pooled RNA from whole developing grainof Chinese Spring harvested at 3,5,7,10,15,25,30 and 35 days postanthesis. The cDNA was cloned into the EcoRI/XhoI site of λ-ZAP II(Stratagene) and the host bacteria used was XL-1Blue (see 2.1 forpreparations of cells).

2.2.2 Rape

(i) cDNA Library from PolyA+RNA

The cDNA library used was generated using the mRNA isolated according tothe method of Logemann et al (1987) from mid stage developing Jet neufrape embryos (harvested at approximately 35 days days post anthesis).The 1st strand synthesis was carried out using poly dT primers accordingto the manufacturers instructions (Amersham International). Theresulting cDNA generated was cloned into the EcoRI/XhoI site of λ-ZAPIIas recommended by the manufacturers (Strater gene). The host bacteriaused was XL-1Blue (see 2.1 for preparation of cells).

ii) Random Primed Library

5 μg of poly A+mRNA from 35 day old (Post anthesis) Jet neuf rape embryowas used for the construction of a random primed cDNA library. Thedouble stranded cDNA was prepared using a 1 in 10 dilution of pd(N)6primers (0.74 μg/ul) according to the instructions provided with TimeSaver™ cDNA synthesis kit (Pharmacia). The library was prepared inλZapII and packaged with Gigapack II Gold packaging extract(Stratagene). The host E.coli strain used was XL-1 Blue (Stratagene).

2.3 Genomic Libraries

The Arabidopsis thaliana library used (a gift from Dr John Cowl, JohnInnes Institute, Norwich) was derived from leaf total DNA in λ FIX IIand the host bacteria used was E.coli KW251 (see 2.1 for preparation ofcells).

2.4 Probe Preparation and Labelling

Plasmid DNA from pA3/DH5α (ICI derived) and pRS1/DH5α (see results for adescription of pRS1) was prepared by the Quagen tip method. Probe forthe screening of Wheat and Rape cDNA libraries was generated by thedigestion of 10 μg pA3 with 20 U EcoRI or Hind III (New EnglandBiolabs). The fragment isolated from the probe was 2.7 and 1.54 kb inlength respectively. Probe for the screening of the Arabidopsis genomiclibrary was generated by a Xho I/Pst I (10 U of each) double digest of10 μg pRS1 to give an isolated fragment size of 1.2 kb. All digests werecarried out in Pharmacia's “one-Phor-All Buffer PLUS” at 37° C. for 3hours. Digests were separated by 1% TAE buffered agarose gelelectrophoresis and the required fragments cut out from the gel. The DNAwas obtained from the gel slice using the method recommended byGeneclean II (Bio 101). DNA concentration was determined byspectrophotometry.

The probes (200-300 ng) were radio-labelled with p³²αdCTP using theMegaprime kit as recommended by the manufacturers (AmershamInternational) to a level of 5×10⁹ dpm/μg. Un-incorporated label wasremoved using Biospin chromatography columns (Biorad).

Just before use for hybridisation the radio-labelled probe was boiledfor 5 minutes and placed on iced water for 2 minutes before being addedto hybridisation buffer at 65° C.

2.5 cDNA Library Primary Screening

For the Wheat cDNA library 300,000 pfu's and the rape random primed andpoly dT primed cDNA library 150,000 pfu's were added to 2 ml ofcompetent XL1-Blue cells (150,000 pfu's/2 ml) mixed and incubated at 37°C. for 20 minutes. The culture was then added to 30 ml top agarose(150,000 pfu's/30 ml) which had been melted and held at 50° C., mixedbriefly and poured onto pre-warmed (37° C.) large LB plates (243×243×18mm). Plates were left at room temperature for 10 minutes and incubatedovernight at 37° C. The plates were finally incubated at 4° C. for 30minutes.

Square sheets of nitrocellulose were carefully placed onto the surfaceof each plate and allowed to soak in for 30 seconds, pealed off andplaced onto 3 mm blotting paper soaked in denaturing buffer (1.5 M NaCl,0.5 M NaOH) for 2 minutes. To neutralise the filters each wassubsequently placed for 5 minutes onto 3 mm paper soaked in neutralisingbuffer (1.5 M NaCl, 0.5 M Tris pH 7.4) and finally for 5 minutes on 3 mmpaper soaked in ×2 SSC. A second lift of 2 minutes was also carried outand treated in the same way. To immobilise the blotted DNA each filterwas placed in a vacuum oven for 30 minutes.

The filters were incubated in pre-hybridisation buffer (50 mls ×6 SSC,×1 Dendhart's, 0.5% SDS, 0.05% sodium pyrophosphate, 50 μg/m¹ herringsperm DNA with constant mixing for 3 hours at 65° C. at which point thebuffer was discarded. The radio-labelled probe (see 2.4) was added to 10ml hybridisation buffer (50 mls ×6 SSC, ×1 Dendhart's, 0.5% SDS, 0.05%Sodium Pyrophosphate, 1 mM EDTA) previously equilibrated to 65° C. Thefilters were incubated with constant mixing for 14 hours at 65° C. andthe hybridisation buffer/probe removed but retained at −20° C. for thesubsequent screens.

To wash off the un-bound probe the filters were washed 4 times with ×1SSC, 0.1% SDS for 30 minutes at 65° C. Filters were air dried andexposed to film overnight. Positive plaques were located and pulled outfrom the plate using the wide end of a 1 ml gilson tip. Only plaquesthat showed up positive on both lifts (30 seconds and 2 minute lifts)were used. The plug was placed into 500 μl SM buffer with 10 μlchloroform and incubated at room temperature for 2 hours with occasionalmixing. The suspension was spun for 5 minutes on a bench top centrifugeand the supernatant containing the pfu's retained.

2.6 Genomic Library Primary Screening

The methods used were as already described (see 2.5) but in a geneticmanipulation isolation unit 2×10⁴ were screened in total on 2 plates.

2.7 cDNA and Genomic Secondary Screening

50-200 pfu's in 200 μl SM buffer were added to 200 μl of competentXL1-Blue cells mixed and incubated at 37° C. for 20 minutes. The culturewas then added to 3 ml melted top agarose at 50° C., mixed briefly andpoured onto pre-warmed 37° C.) small LB plates (850 mm diameter). Plateswere held at room temperature for 10 minutes and incubated overnight at37° C. The plates were finally incubated at 4° C. for 30 minutes.

Pre-hybridisation and hybridisation was carried out in the same way asthat in the primary screen (see 2.5), using the same probe/hybridisationbuffer boiled for 5 minutes before use.

The procedure for lifting, preparing, probing, washing and exposing thenitro-cellulose filters was essentially the same as that alreadydescribed (see 2.5).

The positive plaques were removed as a plug using the wide end of a 200μl Gilson tip, placed into 500 μl SM buffer with 10 μl chloroform andincubated at room temperature for 2 hours with occasional mixing. Thesuspension was spun for 5 minutes on a bench top centrifuge and thesupernatant containing the pfu's retained.

2.8 cDNA and Genomic Tertiary Screening

The method was essentially the same as that for the secondary screen(see 2.7) using only 10-20 pfu's per plant. Exposure of thenitrocellulose filters was only required for 2 hours in this instance.

2.9 Isolation of DNA from Positive Plaques

Plasmid rescue for cDNA clones was carried out as described by theStratagene protocol for “in vivo excision of pSK from λ-ZAPII clones”.The DNA from the pSK derived clones was prepared in large quantitiesusing the Quagen tip method.

2.10 Preparation of Genomic DNA from Positive Plaques

One positive plaque was removed from a plate of the positive pfu's fromthe tertiary screen and incubated with 500 μl fresh KW 251 cells (see2.1 for method of cell preparation) at 37° C. for 20 minutes. Pre-warmedLB media (50 ml at 37° C.) was added in addition to 500 μl M MgSO₄ andincubated with mild shaking at 37° C. for 5-7 hours. Following the 5-7hours, 250 μl Chloroform was added to the culture and incubated for afurther 15 minutes at 37° C. Cell debris was spun out at 10,000 g andDNase/RNase added to the supernatant to a final concentration of 1 μgml⁻¹ and further incubation at 37° C. for 30 minutes. 5 g PolyethyleneGlycol 8000/3.2 g NaCl was added slowly to the supernatant at 4° C.overnight with constant stirring.

The resultant suspension was pelleted at 10,000 g (4° C.) and taken upin 5 ml 20 mM Tris-HCl pH 7.4/100 mM NaCl/10 mM MgSO₄. The solution wasthen subjected to 3-5 chloroform extraction's and 3-5 1:1Phenol:Chloroform extraction. To precipitate the DNA an equal volume ofisopropanol (−20° C.) was added and left on ice for 30 minutes. Theprecipitated DNA was pelleted at 10,000 g and washed in 70% Ethanol(−20° C.) before being pelleted again. The DNA was resuspended in 300 μlT₁₀E₁ buffer.

Subcloning was carried out according to the method used by Sambrook etal (1989).

2.11 Sequencing of DNA Clones

Sequencing was carried out by the manufacturer's recommended methods forthe machine used (Applied Biosystems Inc 373A DNA sequencer). Bothforward and reverse primers (−21 m13 and M13RP1) were used initially forall clones. Oligonucleotides (20 mers) were generated and used tofurther sequence pRS1 (rape ACCase clone). pK111 (Wheat ACCase clone)was subjected to nested deletions by the recommended method (Pharmacia,“d.s. Nested Deletion Kit”) and sequenced by a combination of forwardand reverse primers and generated oligonucleotide priming. Computeranalysis of DNA sequence was carried out using the SEQNET package fromthe SERC facility at Daresbury and DNA Strider.

3. Northern Blot Analysis

Poly A+mRNA was prepared from either 5 g young leaf or 5 g embryosharvested at 15, 22,29, 36, 42 and 49 days post anthesis using therecommended procedure (Pharmacia mRNA purification kit). 1-5 ug wasloaded on to a 1% formamide/formaldehyde agarose gel forelectrophoresis. The Northern blot procedure was as described previouslyElborough et al 1994).

4. Southern Blot Analysis

Total DNA isolated from rape and Arabidopsis leaves (10 ug and 2ug/digestion respectively) was digested with EcoR1, HindIII and BamHIseparately for 8 Hrs. The DNA was separated by TAE agaroseelectrophoresis, blotted and hybridised to radiolabelled probe asdescribed by Sammbrook et al.

RESULTS

1.1 Partial Purification of ACCase from Wheat Germ

Partial purification of Wheat ACCase was carried out essentially usingthe method previously described by Egin-Buhler et al (1980) with severalmodifications.

All operations were carried out at 4° C. unless otherwise stated. Allbuffers used contained 14 mM β-mercaptoethanol and 0.3 mM EDTA.

6×25 g of dry Avalon Wheat germ was ground in a coffee grinder for 15secs. 200 ml 100 mM Tris-HCl pH 7.5 was added to each and polytroned onfull speed for 1 minute. The homogenate was stirred for 15 minutes andspun at 20,000 g. The supernatant was stirred with 25 g wet weight Dowex50 previously equilibrated with 100 mM Tris-HCl pH 7.5 for 15 minutes.The suspension was filtered through cheese cloth and 10%Polyethyleneimine at pH 7.5 added to 0.03% w/v dropwise whilst stirring.After 15 minutes the suspension was spun again at 20,000 g. Powdered(NH₄)₂SO₄ was added to a final saturation of 60% and stirred for 1 hour.After spinning at 20,000 g the pellets were resuspended in 100 ml 100 mMTris-HCl pH 7.5/100 mM NaCl. The supernatant was dialysed for 1 houragainst 5 litres 100 mM Tris-HCl pH 7.5/100 mM NaCl and subsequentlyovernight with fresh buffer (5 litres). Powdered (NH₄)₂SO₄ was added toa final saturation of 25% and stirred for 1 hour spun at 20,000 g andthe supernatant brought up to 70% saturation. After centrifugation theresulting pellet was resuspended in 50 ml 20 mM Tris-ECl pH 7.5, 20 mMNaCl and dialysed with 3×1 hour changes against 5 litres 20 mM Tris-HClpH 7.5, 20 mM NaCl/20% glycerol. The resultant suspension was diluted toa conductivity of <4.3×10⁻³ cm⁻¹ and stirred slowly with 150 ml ofpre-equilibrated Q-sepharose (in 20 mM Tris-Hcl pH 7.5 20 mM NaCl/20%glycerol) for 2 hours. The unbound protein was removed using a sinteredglass funnel and the matrix washed with 10 volumes of 20 mM Tris-HCl pH7.5, 20 mM NaCl/20% glycerol. The slurry was packed into a 10 cmdiameter Pharmacia column. Protein was eluted from the column using agradient of 60-500 mM NaCl/20 mM Tris-HCl pH 7.5/20% glycerol (see FIG.1A for elution profile) at 100 ml hr⁻¹ collecting approx 9 ml fractions.Every other fraction was assayed for ACCase activity, the most activefractions pooled and brought to 50% (NH₄)₂SO₄ saturation. The pelletafter centrifugatior was taken up in a minimal volume (approx 100 ml) of20 mM Tris-HCl pH 7.5, 5 mM MgCl, 20% glycerol to give >4.6×10⁻³ cm⁻¹conductivity. This was incubated with 100 ml pre-equilibratedBlue-sepharose (in 20 mM Tris-HCl, pH 7.5/5 mM MgCl/20% glycerol) withmixing for 2 hours. The matrix was washed with 10 volumes of 20 mMTris-HCl pH 7.5/5 mM MgCl/20% glycerol using a sintered glass funnel.The washed matrix was packed into a 10 cm diameter Pharmacia column andthe protein eluted from the column with a 60-500 mM NaCl/20 mm Tris-HClpH 7.5/5 mM MgCl/20% glycerol gradient (see FIG. 1B for elution profile)at 100 ml 1 hour taking 9 ml fractions. The pooled active fractions(post-Blue-sepharose material) were stored frozen at −70° C.

1.2 Identification of Approx. 220 kDa Protein as Biotin Containing

The dominant 220 kDa a band in the post Blue-sepharose material wasidentified as ACCase by both its ability to change mobility during SDSPAGE in the presence of streptavidin and its estimated molecular weight(Egin-Buhler et al. (1980). SDS PAGE ×5 loading buffer (5 μl) was addedto 20 ul post Blue-sepharose material, boiled at 100° C. for 2 mins. and1 μl of a 5 mM Steptavidin stock added immediately. The solution wasincubated at 650° C. for 5 mins. and loaded onto an SDS PAGE gel next tomyosin (Mr 200 kDa) and untreated post Blue-sepharose material samplefor comparison (see FIG. 2A). Streptavidin clearly reduced the mobilityof the 220 kDa band, indicating that it is biotin containing. The onlyknown biotin enzyme with a MW of 220 kDa is ACCase.

1.3 Generation and Sequencing of Wheat ACCase Peptides

A sample of post-Blue sepharose material estimated to contain approx.400 pM (80 μg) of ACCase, as determined by comparison with knownconcentration standards, was loaded onto an SDS PAGE prep gel (see 1.3for method and FIG. 2B for appearance of sample). The running buffer wasfresh and had a reduced level of SDS (0.035% SDS). Chromaphor green(Promega) was added at 1:1000 dilution to the upper tank duringelectrophoresis to allow the visualisation of protein. the ACCaseprotein band at approx. 220 kDa was cut out of the gel, frozen andstored at −20° C. overnight. The gel slices were trimmed of excessacrylamide and loaded on to one well of a 3 mm thick large BioradProtean gel. The gel slices once loaded were overlaid withEndoproteinase LysC (Promega) at 6.5% protein concentration in 50%glycerol/0.125M Tris pH 6.8,/0.1%. SDS/3% B-mercaptoethanol/0.005%Bromophenol Blue. The gel was run until the protein was at the stackerinterface at which point electrophoresis was stopped for 1 hr at roomtemperature. Electrophoresis was resumed until the dye front reached thebottom of the gel. Peptides were semi-dry blotted into ProBlot (AppliedBiosystems Inc.) according to manufacturers instructions. RapidCoomassie staining of the blot (according to ProBlot instructions)identified peptide fragments which were excised from the membrane andloaded onto an ABI 477A pulse liquid protein sequencer. Sequence datawas obtained at an amino acid level of 10-20 pM (see FIG. 3).

Sequence data was obtained for 4 peptides, yielding stretches ofN-terminal amino acid sequence of 17, 18, 9 and 20 amino acids (FIG. 3).

2. ACCase Clone Isolation and Sequencing

2.1 Wheat ACCase cDNA

A wheat cDNA library was probed with a 2.7 kb EcoR1 fragment, and a 1.54kb HindIII fragment of the maize partial cDNA clone pA3 which contains4.5 kb of the 3′ maize ACCase. This yielded a 1.85 kb clone insertedbetween the Eco R1 and XhoI site in the multi cloning cassette of pSK.The DNA was recovered by plasmid rescue in the host strain DH5α. Thisclone was denoted pK111.

The nucleotide sequence data of this partial cDNA, with the derivedamino acid sequence from the three reading frames is shown in FIG. 4.

FIG. 4 also shows that sections of pK111 show complete homology with theamino acid sequence of the 4 peptides isolated from the purified wheatgerm enzyme, providing good evidennce that the cDNA does indeed code forwheat embryo ACCase.

A dot matrix comparison of the deduced amino acid sequence from thelargest open reading frame against that of the maize ACCase is presentedin FIG. 5. pK111 showed 82.33% homology with the maize cDNA at thenucloetide level and 88.17% similarity/78.44% identity at the amino acidlevel.

In addition the deduced amino acid data of the wheat cDNA showed largehomologous regions with the known sequences of rat (62%) and yeast (62%)ACCase.

2.2 Isolation of a Partial Rape ACCase cDNA Encoding theTranscarboxylase Domain

Although ACCase has been purified from rape embryo, the amounts obtainedwere not amenable to protein sequencing. To study ACCase at the sequencelevel we needed to isolate its cDNA. A rape embryo derived poly dTprimed λZapII library was screened with the partial wheat ACCase cDNApreviously isolated. A hybridising cDNA of 2.5 kb was taken throughthree rounds of screening and plasmid rescued (pRS1). The clone wasfully sequenced in both directions by a combination of nested deletionsand dye primer sequencing. The cDNA sequence has been submitted to EMBL(Accession no. X77382). The predicted amino acid sequence from thelargest open reading frame is shown in FIG. 6. Dot matrix analysis ofthe cDNA with previously described ACCase sequences showed it to be apartial clone of ACCase corresponding to the transcarboxylase domain(FIG. 7). The predicted amino sequence of the rape clone showed sequenceidentity/similarity levels of approximately 44/61% with the yeast(Al-Feel et al, 1992), rat (Lopez-Casillas et al, 1988) algae (Roesslerand Ohlrogge, 1993) and the wheat ACCase cDNA pK111. Since the mRNAcontaions a polyA tail and was obtained from the poly A fraction it isprobable that the ACCase cDNA isolated was nuclear encoded.

2.3. Isolation of the Arabidopsis ACCase Genomic Clone and Further RapecDNA Cloning

The average insert size of our rape poly dT primed cDNA library,described above, was approximately 2-2.5 kb. Therefore it was unlikelythat the library would contain much more 5′ cDNA. To obtain more 5′sequence a random primed library from rape embryo mRNA was constructed.Having made a suitable library there were two strategies available forcloning more 5′ cDNA i) screen using the 5′ region of our cDNA, or; ii)screen using 5′ probes from a genomic clone. We chose the second option.The strategy was to clone the ACCase genomic gene, identify the openreading frames by sequence comparison and generate specific probes bythe use of PCR. Since Arabidopsis is related to rape and has a smallergenome we chose to obtain the genomic clone from Arabidopsis. Previousdata from this laboratory had shown that Arabidopsis DNA sequences arehighly homologous to those of rape (data not shown). Screening a λ FixIIArabidopsis genomic library with a 1.2 kb Xho1/Pst1 fragment of the rapeACCase cDNA pRS1 yielded two independent genomic clones which hybridisedstrongly to the pRS1 ACCase probe. These were denoted λAYE4 and λAYE8.λAYE8 was subcloned to produce two plasmids:pKLU81, a 5.3 kb subclone inthe EcoR1 site of pGEM 3ZF+; and pKLS2, which was excised from the λclone by a partial Sa11 digest and subcloned into pSK+.

The pKLU81 subclone, considered to be a partial length genomic clone,was partially sequenced-from the 5′ and 3′ ends. Therefore two sets ofdata are presented for the 5′ and 3′ sequences from the same clone. Thenucleotide sequences, with the derived amiono acid sequences from the 3reading frames are shown in FIGS. 8 and 9. A data base search(Swissprot) using the derived amino acid sequence from the 5′ 0.56 kbDNA sequence showed 40% identity with chicken and rat ACCase (FIG. 10).

The genomic clone (pKLS2) was extensively subcloned through acombination of EcoRI/SalI/XbaI/HindIII digests, and partially sequencedby both Dye primer and Dye terminator chemistry. We found thatintron-exon boundaries could not be allocated without cDNA data. Wetherefore opted to sequence only enough of the genomic clone to allowgeneration of open reading frame probes for cDNA screening. The fullsequence data obtained is shown schematically in FIG. 11A (hatched areasA,Aii,B,C,D,E and F) and has been deposited with the EMBL data base(accession no's X77375-X77381).

To map the ACCase activity domain order, within the genomic clone, theopen reading frame sequences from the different sequenced areas werecompared with the first two domains of the full length yeast cDNA (FIG.11Bi and ii), and the rape transcarboxylase domain (FIG. 11Biii).Homology was sufficient to allow us to assign the same order of domainsto the Arabidopsis gene as that of yeast ACCase shown in FIG. 11A ie:[Biotin carboxylase-Biotin binding-Transcarboxylase].

Sequence data from an open reading frame at the 5′ end of the genomicclone (area Aii) showed a marked homology (49.5/64% identity/similarityat the derived amino acid level) with the 5′ region of yeast ACCase (seeFIG. 11Bi). The 3′ end of the cloned genomic fragment (19 kb) wassequenced and shown to be homologous to the 3′ end of the rape 2.5 kbcDNA clone isolated from the poly d'T primed mRNA library (FIG. 11Biii).Since we had approximately 1.3 kb 5′ to area Aii we reasoned that it waslikely that pKLS2 was the full length genomic clone. The pKLU 81subclone was a partial length genomic clone corresponding to a portionof the sequence of pKLS2.

Since the Arabidopsis genomic clone showed a high degree of homology tothe rape cDNA isolated (86% identity in the exons of areas E and F) itwas clear that the genomic clone could be used to isolate further-rapecDNA's. We generated a specific probe via PCR of area C within thegenomic clone and used it to screen the random primed library generatedfrom rape embryo mRNA. Two cDNA clones (pRS8 and pRS6 containing 2.0 kband 1.1 kb cDNA respectively) were isolated and sequenced. The cDNA fromeach was shown to overlap. The full combined derived amino acid sequence(pRS8/6 2.38 kb cDNA size) is presented in FIG. 12Ai (EMBL accession noX77374). The sequence analysis of the clones showed significant homologywith that of yeast (39/58% identity/similarity), rat (38/59%identity/similarity) and algal (34/54% identity/similarity) ACCase.Within the cDNA sequence is the highly conserved biotin binding site[Val-Met-Lys-Met], shown in FIG. 12Ai as the underlined region. Directcomparison with yeast biotin binding site is shown in FIG. 12Aii.Interestingly the sequence also showed homology at it's 5′ end with the3′ portion of the yeast biotin carboxylase domain. This datademonstrated that the domain order in rape [Biotin carboxylase-Biotinbinding-Transcarboxylase] is consistent with the domain assignment ofArabidopsis.

3. Southern Blot Analysis

Since it was not known how many genes for ACCase are present in rape andArabidopsis, total DNA was analysed by Southern blotting. Both rape andArabidopsis total DNA was digested with three separate restrictionenzymes and blotted. The Arabidopsis genomic clone indicated that ACCasegenes would most likely be relatively large. The size dictated that itwas not possible, using partial cDNA's as probes, to gain an accurateestimate of the gene copy number by Southern blot. The blot wastherefore hybridised to the full Arabidopsisg genomic clone 19 kb)labelled by random primed labelling. The sum of the Arabidopsis bandsthat hybrised to the probe was approximately 20 kb (FIG. 13). Since thegenomic clone is approximately 19 kb, and showned a similar pattern whendigested with the same enzymes (results not shown), we deduced thatthere is only the one gene present in Arabidopsis. Although the rapeprofile is more complicated it can be seen that it consists of arelatively small gene family (see FIG. 13).

4. Northern Blot Analysis

The expression of ACCase during rape embryonic development was examinedby Northern blotting using the 2.5 kb rape cDNA clone as probe. Theblotrs contained 5 ug rape poly A+mRNA prepared from a set of stagedembryos taken from Brassica napus Jet Neuf at 15, 22, 29, 35,42 and 49days post-anthesis. Embryos taken from the same seed set were alsoanalysed for oil content to monitor development. The oil content data ispresented (expressed as fatty acid/mg seed) graphically in FIG. 14A. TheNorthern blot was hybridised separately to three successive probes andstripped after each in preparation for the next probe. The three probesused-were embryo derived cDNAs for enoyl reductase (1.15 kb), βketoreductase (1.185 kb) and ACCase (2.5 kb). All three cDNAs were highlyexpressed in seed with maximum expression being coincidental at 29 dayspost-anthesis (FIG. 14A). However it appears that the initial onset ofmRNA production occurs in the order enoyl reductase, βketo reductase andACCase. The profile of all three genes expression during embryogenesiswas reproducable in individually probed blots with peak expressionoccuring at 29 days. The sizes of the hybridising bands were 1.65, 1.7and 7.5 kb respectively as determined by size markers run on the sameagarose gel used for the blot. The level of the ACCase mRNA wasrelatively lower than that of enoyl reductase and βketoreductase. Thismay be in part due to the successive stripping of the blot anddegradation of the large 7.5 kb message during handling.

A Northern blot comparison of ACCase expression in 29 days post-anthesisembryo and young leaf, using the embryo derived 2.5 kb cDNA as a probeis shown in FIG. 14B. The 7.5 kb band that hybridises was approximatelyfive times more abundant in seed than in leaf, as might be expected forACCase. The size of the full length mRNA (7.5 kb) was consistent withthe known size of the full length mRNA for both maize and wheat ACCase.

References

Al-Feel, W., Chirala, S. S., Wakil, S. J. (1992) Proc. Natl. Acad. Sci.89, 4534-4538.

Egin-Buhler, B et al (1980) Arch Biochem Biophys 203, 90-100.

Elborough et al (1994) Plant Mol. Biol. 24, 21-34.

Hellyer, A. et al (1986) Biochem Soc Trans 14, 565-568.

Laemmli (1970) Nature (Lond) 227, 680-685.

Logemann, J et al (1987) Anal. Biochem. 163, 16-20

Lopez-Casillas, F. et al (1988) Proc. Natl. Acad. Sci. 85, 5784-5788.

Roessler, P. G. and Ohlrogge, J. B. (1993) J. Biol Chem 268, 19254-19259

Sammbrook, J. (1989) “Molecular Cloning: A laboratory Manual” 2ndedition, CSH Laboratory Press

32 17 amino acids amino acid linear peptide NO internal Avena sativaAvalon 1 Met Ala Thr Asn Gly Val Glu Xaa Leu Thr Val Ser Asp Asp Leu Glu1 5 10 15 Gly 17 amino acids amino acid linear peptide YES internalAvena sativa Embryo pK111 2 Met Ala Thr Asn Gly Val Val His Leu Thr ValSer Asp Asp Leu Glu 1 5 10 15 Gly 18 amino acids amino acid linearpeptide NO internal Avena sativa 3 Leu Gly Gly Ile Pro Val Gly Val IleAla Val Glu Thr Gln Thr Xaa 1 5 10 15 Asp Gln 18 amino acids amino acidlinear peptide YES internal Avena sativa Embryo pK111 4 Leu Gly Gly IlePro Val Gly Xaa Ile Ala Val Glu Thr Gln Thr Met 1 5 10 15 Met Gln 9amino acids amino acid linear peptide NO internal Avena sativa 5 Asn ValLeu Glu Pro Gln Gly His Leu 1 5 9 amino acids amino acid linear peptideYES internal Avena sativa embryo pK111 6 Asn Val Xaa Glu Xaa Gln Gly LeuIle 1 5 20 amino acids amino acid linear peptide NO internal Avenasativa 7 Ser Ile Glu Ala Arg Lys Lys Gln Leu Leu Pro Leu Tyr Thr Gln Ile1 5 10 15 Ala Ile Arg Phe 20 20 amino acids amino acid linear peptideYES internal Avena sativa pK111 embryo 8 Ser Ile Glu Pro Arg Lys Lys GlnLeu Leu Pro Leu Tyr Thr Gln Ile 1 5 10 15 Ala Val Arg Phe 20 1926 basepairs nucleic acid double linear cDNA to mRNA NO NO Avena sativa Avalonembryo pK111 9 GAGAACATAC ATGGAAGTGC TGCTATTGCC AGTGCCTATT CTAGGGCCTATGAGGAGACA 60 TTTACGCTTA CATTTGTGAC TTGACGGACT GTTGGAATAG GAGCATATCTTGCTCGACTT 120 GGCATACGGT GCATACAGCG TACTGACCAG CCCATTATCC TAACCGGGTTCTCTGCTTTG 180 AACAAGCTTC TTGGCCGGGA AGTGTACAGC TCCCACATGC AGTTGGGTGGCCCCAAAATT 240 ATGGCGACAA ACGGTGTTGT CCATCTGACA GTTTCAGATG ACCTTGAAGGTGTGTCTAAT 300 ATATTGAGGT GGCTCAGCTA TGTTCCTGCC AACATTGGTG GACCTCTTCCTATTACAAAA 360 TCTTTGGACC CACCTGACAG ACCCGTTGCA TATATCCCTG AGAATACATGTGATCCTCGT 420 GCAGCCATCA GTGGCATTGA TGATAGCCAA GGGAAATGGT TGGGGGGCATGTTCGACAAA 480 GACAGTTTTG TGGAGACATT TGAAGGATGG GCGAAGTCAG TAGTTACTGGCAGAGCGAAA 540 CTCGGAGGGA TTCCGGTGGG TGTNATAGCT GTGGAGACAC AGACTATGATGCAGCTCATC 600 CCTGCTGATC CAGGGCAGCT TGATTCCCAT GAGCGGTCTG TTCCTCGTNCTGGGCAAGTN 660 TGGTTTCCAN ATTNANCTAC TAAGACAGCT CAAGCAATGC TGGACTTCAACCGTNAAGGA 720 TTACCTCTNT TCATCCTTGC NAACTGGAGA GGCTTCTCTG GTGGGCAAAGAGATCTTTTT 780 AAAGGAATCC TTCAGGCTGG GTCAACAATT GTTGAGAACC TTAGGACATACAATCAGCCT 840 GCCTTTGTAT ATATCCCCAA GGCTGCAGAG CTACGTGGAG GGGCTTGGGTCGTGATTGAT 900 AGCAAGATAA ATCCAGATCG ATTTGAGTTC TATGCTGAGA GGACTGCAAAGGGTAATGTT 960 CTNGAACCNC AAGGGTTGAT TGANATCAAN TTCAGGTCAG AGGAACTCCAAGAGTGCATG 1020 GGCAGGGTTG ACCCAGAATT GATAAATCTG AAGGCAAAAC TCCTGGGAGCAAAGCATGAC 1080 AATGGAAGTC TATCTGAGTC AGAATCCCTT CAGAAGAGCA TAGAACCCCGGAAGAAACAG 1140 TTGTTGCCTT TGTATACTCA AATTGCGGTG CGGTTTGCTG AATTGCATGACACTTCCCTT 1200 AGAATGGCTN CTAAGGGTGT GATTAAGAAG GTTGTAGACT GGAAAGATTCTAGGTCTTTC 1260 TTCTACAAGA GATTACGGAG GAGGATATCC GAGGACGTTC TTGCAAAGGAAATTAGAGGT 1320 GTAAGTGGCA AGCAGTTCTC TCACCAATCA GCAATCGAGC TGATCCAGAAATGGTACTTG 1380 GCTTCTAAGG GAGCTGAAGC AGCAAGCACT GAATGGGATG ATGACGATGCTTTTGTTGCC 1440 TGGAGGGAAA ACCCTGAAAA CTACCAGGAG TATATCAAAG AACTTAGGGCTCAAAGGGTA 1500 TCTCAGTTGC TCTCAGATGT TGCAGACTCC AGTCCAGATC TAGAAGCCTTGCCACAGGGT 1560 CTTTCTATGC TACTAGAGAA GATGGATCCC TCAAGGAGAG CACAGTTTGTTGAGGAAGTC 1620 AAGAAAGTCC TTAAATGATC AGATGATACC AACGCATCCA ATTCAGAATGTGCATGATAT 1680 CGGTTTCTCT TGAAGTACAT ATATAGANGG ATACTATTCG GCTGTAACCGACCATAGCTG 1740 ATCTGAGTCA ACCATTATTT TGTAAAACTT TTTTGCGGTC TTCTCTGTTATTCGAGGCAA 1800 AACTTGTTTT CGGACGGCTC CGAATGGTTG ATGAGTGTAG TTGGAAAAAAAGCGGCCGGA 1860 ATTNCTGCAG CCCGGGGGAT CCNCTAGTTC TAGAGCGGCC GCACCGGGTTGGAGNTCCAG 1920 TTTTTT 1926 642 amino acids amino acid linear proteinYES N-terminal Avena sativa Avalon embryo pK111 Region 81..97 Region181..198 Region 319..327 Region 373..392 10 Glu Asn Ile His Gly Ser AlaAla Ile Ala Ser Ala Tyr Ser Arg Ala 1 5 10 15 Tyr Glu Glu Thr Phe ThrLeu Thr Phe Val Thr Xaa Arg Thr Val Gly 20 25 30 Ile Gly Ala Tyr Leu AlaArg Leu Gly Ile Arg Cys Ile Gln Arg Thr 35 40 45 Asp Gln Pro Ile Ile LeuThr Gly Phe Ser Ala Leu Asn Lys Leu Leu 50 55 60 Gly Arg Glu Val Tyr SerSer His Met Gln Leu Gly Gly Pro Lys Ile 65 70 75 80 Met Ala Thr Asn GlyVal Val His Leu Thr Val Ser Asp Asp Leu Glu 85 90 95 Gly Val Ser Asn IleLeu Arg Trp Leu Ser Tyr Val Pro Ala Asn Ile 100 105 110 Gly Gly Pro LeuPro Ile Thr Lys Ser Leu Asp Pro Pro Asp Arg Pro 115 120 125 Val Ala TyrIle Pro Glu Asn Thr Cys Asp Pro Arg Ala Ala Ile Ser 130 135 140 Gly IleAsp Asp Ser Gln Gly Lys Trp Leu Gly Gly Met Phe Asp Lys 145 150 155 160Asp Ser Phe Val Glu Thr Phe Glu Gly Trp Ala Lys Ser Val Val Thr 165 170175 Gly Arg Ala Lys Leu Gly Gly Ile Pro Val Gly Xaa Ile Ala Val Glu 180185 190 Thr Gln Thr Met Met Gln Leu Ile Pro Ala Asp Pro Gly Gln Leu Asp195 200 205 Ser His Glu Arg Ser Val Pro Arg Xaa Gly Gln Xaa Trp Phe ProXaa 210 215 220 Xaa Xaa Thr Lys Thr Ala Gln Ala Met Leu Asp Phe Asn ArgXaa Gly 225 230 235 240 Leu Pro Xaa Phe Ile Leu Xaa Asn Trp Arg Gly PheSer Gly Gly Gln 245 250 255 Arg Asp Leu Phe Lys Gly Ile Leu Gln Ala GlySer Thr Ile Val Glu 260 265 270 Asn Leu Arg Thr Tyr Asn Gln Pro Ala PheVal Tyr Ile Pro Lys Ala 275 280 285 Ala Glu Leu Arg Gly Gly Ala Trp ValVal Ile Asp Ser Lys Ile Asn 290 295 300 Pro Asp Arg Phe Glu Phe Tyr AlaGlu Arg Thr Ala Lys Gly Asn Val 305 310 315 320 Xaa Glu Xaa Gln Gly LeuIle Xaa Ile Xaa Phe Arg Ser Glu Glu Leu 325 330 335 Gln Glu Cys Met GlyArg Val Asp Pro Glu Leu Ile Asn Leu Lys Ala 340 345 350 Lys Leu Leu GlyAla Lys His Asp Asn Gly Ser Leu Ser Glu Ser Glu 355 360 365 Ser Leu GlnLys Ser Ile Glu Pro Arg Lys Lys Gln Leu Leu Pro Leu 370 375 380 Tyr ThrGln Ile Ala Val Arg Phe Ala Glu Leu His Asp Thr Ser Leu 385 390 395 400Arg Met Ala Xaa Lys Gly Val Ile Lys Lys Val Val Asp Trp Lys Asp 405 410415 Ser Arg Ser Phe Phe Tyr Lys Arg Leu Arg Arg Arg Ile Ser Glu Asp 420425 430 Val Leu Ala Lys Glu Ile Arg Gly Val Ser Gly Lys Gln Phe Ser His435 440 445 Gln Ser Ala Ile Glu Leu Ile Gln Lys Trp Tyr Leu Ala Ser LysGly 450 455 460 Ala Glu Ala Ala Ser Thr Glu Trp Asp Asp Asp Asp Ala PheVal Ala 465 470 475 480 Trp Arg Glu Asn Pro Glu Asn Tyr Gln Glu Tyr IleLys Glu Leu Arg 485 490 495 Ala Gln Arg Val Ser Gln Leu Leu Ser Asp ValAla Asp Ser Ser Pro 500 505 510 Asp Leu Glu Ala Leu Pro Gln Gly Leu SerMet Leu Leu Glu Lys Met 515 520 525 Asp Pro Ser Arg Arg Ala Gln Phe ValGlu Glu Val Lys Lys Val Leu 530 535 540 Lys Xaa Ser Asp Asp Thr Asn AlaSer Asn Ser Glu Cys Ala Xaa Tyr 545 550 555 560 Arg Phe Leu Leu Lys TyrIle Tyr Arg Xaa Ile Leu Phe Gly Cys Asn 565 570 575 Arg Pro Xaa Leu IleXaa Val Asn His Tyr Phe Val Lys Leu Phe Cys 580 585 590 Gly Leu Leu CysTyr Ser Arg Gln Asn Leu Phe Ser Asp Gly Ser Glu 595 600 605 Trp Leu MetSer Val Val Gly Lys Lys Ala Ala Gly Ile Xaa Ala Ala 610 615 620 Arg GlyIle Xaa Xaa Phe Xaa Ser Gly Arg Thr Gly Leu Glu Xaa Gln 625 630 635 640Phe Phe 765 amino acids amino acid single linear protein NO NO internalBrassica napus 11 Ala Arg Gly Arg Asn Ser Leu Ile Tyr His Ser Ile ThrLys Lys Gly 1 5 10 15 Pro Leu His Gly Thr Gln Ile Asn Asp Gln Tyr LysPro Leu Gly Tyr 20 25 30 Leu Asp Arg Gln Arg Leu Ala Ala Arg Arg Ser AsnThr Thr Tyr Cys 35 40 45 Tyr Asp Phe Pro Leu Ala Phe Glu Thr Ala Leu GluGln Phe Gly His 50 55 60 Tyr Asn Asn Arg Glu Leu Arg Asn His Ala Arg ValLeu Leu Ser Val 65 70 75 80 Leu Lys Ser Leu Tyr Ser Pro Ile Ser Glu GlyThr Ser Leu Met Pro 85 90 95 Val Glu Arg Ser Pro Gly Leu Asn Glu Phe GlyMet Val Ala Trp Ser 100 105 110 Leu Glu Met Ser Thr Pro Glu Phe Pro MetGly Arg Lys Leu Leu Ile 115 120 125 Val Ala Asn Asp Val Thr Phe Lys AlaGly Ser Phe Gly Pro Arg Glu 130 135 140 Asp Ala Phe Phe Leu Ala Val ThrGlu Leu Ala Cys Pro Lys Lys Leu 145 150 155 160 Pro Leu Ile Tyr Leu AlaPro Asn Ser Gly Ala Arg Leu Gly Val Ala 165 170 175 Glu Glu Ile Lys AlaCys Phe Lys Val Gly Trp Ser Asp Glu Val Ser 180 185 190 Pro Glu Asn GlyPhe Gln Tyr Ile Tyr Leu Ser Pro Glu Asp His Ala 195 200 205 Arg Ile GlySer Ser Val Ile Ala His Glu Ile Lys Leu Pro Ser Gly 210 215 220 Glu ThrArg Trp Val Ile Asp Thr Ile Val Gly Lys Glu Asp Gly Ile 225 230 235 240Gly Val Glu Asn Leu Thr Gly Ser Gly Pro Ile Ala Gly Ala Tyr Ser 245 250255 Arg Ala Tyr Asn Glu Thr Phe Thr Leu Thr Phe Val Ser Gly Arg Thr 260265 270 Val Gly Ile Gly Ala Tyr Leu Ala Pro Leu Gly Met Arg Cys Ile Gln275 280 285 Arg Leu Asp Gln Pro Ile Ile Leu Thr Gly Phe Ser Thr Leu AsnLys 290 295 300 Leu Leu Gly Arg Glu Val Tyr Ser Ser His Met Gln Leu GlyGly Pro 305 310 315 320 Lys Ile Met Gly Thr Asn Gly Val Val His Leu ThrVal Ser Asp Asp 325 330 335 Leu Glu Gly Val Ser Ala Ile Leu Asp Trp LeuSer Tyr Ile Pro Ala 340 345 350 Tyr Val Gly Gly Pro Leu Pro Val Leu AlaPro Leu Asp Pro Pro Asp 355 360 365 Arg Thr Val Glu Tyr Val Pro Glu AsnSer Cys Asp Pro Arg Ala Ala 370 375 380 Ile Ala Gly Val Asn Asp Asn ThrGly Lys Trp Leu Gly Gly Ile Phe 385 390 395 400 Asp Lys Asn Ser Phe IleGlu Thr Leu Glu Gly Trp Ala Arg Thr Val 405 410 415 Val Thr Gly Arg AlaLys Leu Gly Gly Ile Pro Val Gly Val Val Ala 420 425 430 Val Glu Thr GlnThr Val Met Gln Ile Ile Pro Ala Asp Pro Gly Gln 435 440 445 Leu Asp SerHis Glu Arg Val Val Pro Gln Ala Gly Gln Val Trp Phe 450 455 460 Pro AspSer Ala Gly Lys Thr Ala Gln Ala Leu Met Asp Phe Thr Arg 465 470 475 480Lys Ser Phe His Cys Leu Ser Leu Arg Thr Gly Glu Gly Phe Gln Val 485 490495 Gly Arg Glu Ile Phe Ser Lys Glu Tyr Phe Arg Gln Val Ala Thr Ile 500505 510 Val Glu Asn Leu Arg Thr Tyr Arg Gln Pro Val Phe Val Tyr Ile Pro515 520 525 Lys Met Gly Glu Leu Arg Gly Gly Ala Trp Val Val Val Asp SerGln 530 535 540 Ile Asn Ser Asp Tyr Val Glu Met Tyr Ala Asp Glu Thr AlaArg Gly 545 550 555 560 Asn Val Leu Glu Pro Glu Gly Thr Ile Glu Ile LysPhe Arg Thr Lys 565 570 575 Glu Met Leu Glu Cys Met Gly Arg Leu Asp ProLys Leu Ile Asp Leu 580 585 590 Lys Ala Arg Leu Gln Asp Pro Asn Gln SerGlu Ala Tyr Thr Asn Ile 595 600 605 Glu Leu Leu Gln Gln Gln Ile Lys AlaArg Glu Lys Leu Leu Leu Pro 610 615 620 Val Tyr Ile Gln Ile Ala Thr LysPhe Ala Glu Leu His Asp Thr Ser 625 630 635 640 Met Arg Met Thr Ala LysGly Val Ile Lys Met Cys Val Glu Trp Ile 645 650 655 Gly Ser Arg Ser PhePhe Tyr Lys Lys Leu Asn Arg Arg Ile Ala Glu 660 665 670 Asn Ser Leu ValLys Asn Val Arg Glu Ala Ser Gly Asp Asp Leu Ser 675 680 685 Tyr Lys SerAla Met Gly Leu Ile Gln Asp Trp Phe Ser Lys Ser Asp 690 695 700 Ile ProLys Gly Lys Glu Glu Ala Trp Thr Asp Asp Gln Val Phe Phe 705 710 715 720Thr Trp Lys Asp Asn Val Ser Asn Tyr Glu Leu Asn Leu Ser Glu Leu 725 730735 Arg Pro Gln Lys Leu Leu Asn Pro Thr Cys Arg Asp Trp Lys Phe Arg 740745 750 Arg Ile Tyr Arg Arg Cys His Lys Asp Leu Pro Ile Phe 755 760 7652536 base pairs nucleic acid double linear cDNA to mRNA NO NO Brassicanapus embryo poly dT primed lambda ZapII pRS1 12 GCACGAGGGA GAAACAGTTTGATTTACCAC TCAATTACCA AGAAGGGACC TTTGCATGGA 60 ACCCAAATCA ATGATCAATATAAGCCACTG GGATATCTTG ACAGGCAACG TCTAGCCGCA 120 AGGAGGAGTA ACACTACATATTGCTATGAC TTCCCGTTGG CATTTGAGAC AGCCTTGGAG 180 CAGTTTGGGC ATTACAACAACCGGGAGTTA AGAAACCATG CAAGGGTACT CTTATCAGTG 240 CTAAAGAGCT TGTATTCTCCAATTTCAGAA GGTACATCTC TTATGCCAGT TGAAAGATCA 300 CCGGGTCTCA ATGAGTTTGGAATGGTGGCC TGGAGCCTAG AGATGTCGAC TCCTGAGTTT 360 CCTATGGGAC GGAAGCTTCTCATAGTCGCC AATGATGTCA CCTTCAAAGC TGGTTCTTTT 420 GGTCCTAGAG AGGACGCGTTTTTCCTTGCC GTGACTGAAC TCGCATGTCC CAAGAAGCTT 480 CCCTTGATTT ACTTGGCACCAAATTCTGGT GCCAGACTCG GAGTAGCTGA AGAAATCAAA 540 GCCTGCTTTA AAGTTGGATGGTCGGATGAA GTTTCCCCCG AAAATGGTTT TCAGTATATA 600 TACCTAAGCC CTGAAGACCATGCAAGGATT GGATCATCTG TCATTGCGCA CGAAATAAAG 660 CTCCCTAGTG GGGAAACAAGGTGGGTGATT GATACAATCG TTGGTAAAGA AGATGGTATT 720 GGTGTAGAGA ATCTAACCGGAAGTGGGCCA ATAGCGGGCG CTTACTCGAG GGCATACAAC 780 GAAACATTTA CTTTGACCTTTGTTAGTGGA AGAACGGTAG GAATTGGTGC TTACCTTGCC 840 CCCCTTGGTA TGCGGTGTATACAGAGACTT GACCAGCCGA TCATATTGAC TGGCTTTTCT 900 ACGCTCAACA AGTTACTTGGGCGTGAGGTC TATAGCTCTC ACATGCAACT TGGTGGCCCG 960 AAAATCATGG GCACAAATGGTGTTGTTCAT CTTACAGTCT CAGATGATCT CGAAGGTGTA 1020 TCAGCGATTC TCGACTGGCTGAGCTACATT CCTGCTTACG TTGGTGGTCC TCTTCCTGTT 1080 CTTGCCCCGT TAGACCCACCGGACAGAACC GTGGAGTACG TTCCAGAGAA CTCTTGCGAC 1140 CCGCGAGCTG CTATAGCTGGGGTTAACGAC AATACCGGTA AATGGCTTGG CGGTATCTTT 1200 GATAAAAATA GCTTTATTGAGACTCTTGAA GGCTGGGCAA GAACGGTAGT GACTGGTAGA 1260 GCTAAACTAG GGGGAATACCTGTAGGAGTT GTTGCGGTTG AGACACAGAC AGTAATGCAG 1320 ATCATCCCAG CAGATCCAGGACAGCTCGAC TCTCATGAAA GAGTGGTTCC ACAGGCAGGG 1380 CAAGTCTGGT TTCCTGATTCTGCGGGCAAG ACAGCTCAAG CGCTCATGGA TTTCACAAGG 1440 AAGAGCTTCC ATTGTTTATCCTTGCGAACT GGAGAGGGTT TTCAGGTGGG CAGAGAGATC 1500 TTTTCGAAGG AATACTTCAGGCAGGTTGCG ACTATTGTAG AAAATCTGAG AACGTATCGG 1560 CAGCCAGTGT TTGTGTACATCCCTAAGATG GGAGAGTTGC GAGGTGGAGC GTGGGTTGTT 1620 GTTGATAGCC AAATAAATTCAGATTATGTT GAAATGTATG CTGATGAAAC TGCTAGGGGG 1680 AATGTGCTTG AGCCAGAAGGAACGATAGAG ATAAAATTTA GAACGAAAGA GATGTTAGAG 1740 TGCATGGGAA GGTTAGACCCGAAGCTAATC GATCTCAAAG CAAGACTGCA AGATCCCAAC 1800 CAAAGTGAGG CTTATACAAATATCGAGCTC CTCCAGCAAC AGATTAAAGC CCGAGAGAAG 1860 CTTCTCTTAC CAGTTTATATCCAAATCGCC ACCAAATTTG CGGAACTTCA CGATACTTCC 1920 ATGAGAATGA CTGCCAAAGGAGTGATCAAA ATGTGTGTGG AGTGGATCGG CTCGAGGTCC 1980 TTCTTCTATA AGAAGCTCAACCGGAGAATT GCTGAGAACT CTCTTGTGAA AAACGTAAGA 2040 GAAGCTTCAG GAGACGACTTATCGTATAAA TCTGCAATGG GTTTAATTCA GGATTGGTTC 2100 TCCAAATCTG ACATTCCAAAGGGGAAAGAA GAAGCTTGGA CAGACGACCA AGTGTTCTTT 2160 ACATGGAAGG ACAACGTTAGTAACTACGAG TTGAATCTGA GCGAATTGAG ACCGCAGAAA 2220 CTGTTGAACC CAACTTGCAGAGATTGGAAA TTCCGTCGGA TCTATCGGCG CTGCCACAAG 2280 GACTTGCCAA TCTTCTAAACAAGGTGGAGC CTTCAAGAAG AGAAGAGCTT GTTGAAGCGC 2340 TACGAAAAGT GTTAGGTTGATGTACAAGAG GTCAAGCTTG TGACCCGAGA AAGATGGTCC 2400 TTTGGTGTTG CTTGTGTCCTACGGTGAAAG AAGCTAGTTG GAAATTAGAT GTGGTCTTTC 2460 TTTCTTAAAT GTGTTGGCCCGAGCTGTAAA TGTTGTTGTA GCGTATAAGT GAGAATTGCG 2520 TAATAATTTA TTCAAC 2536565 base pairs nucleic acid double linear DNA (genomic) NO NOArabidopsis thaliana lambda FixII type pKLU81 13 CTCTCTGGCA AATCCCTGGTATAATCTACG TCCTTATTTC TTACAGGCAG CGGTTCCTCT 60 TCTTTATCCA TGCACACGAATAATGTACTG TCTGTTTCTC TTTAATTTCG TAGAGATAAG 120 ACGGTTCTAT GGAATAGAACATGGTGGAGG TTATGATTCT TGGCGAAAAA CATCTGTTGT 180 AGCCTTCCCT TTTGATTTTGATAAAGCTCA ATCTATAAGG CCAAAAGGTC ATTGTGTGGC 240 TGTACGTGTG ACAAGTGAGGTATCCTGATG ACGGGTTCAA ACCAACCAGC GGTAGAGTTC 300 AGGTAATGTG ATATCTGTGGAATGCAAAGT GAAAGTTCAT TCACTGAGGA ACTCTGTGGG 360 GTAACACTTG TATGAACTTGCAACAGGNGT TGAGTTTTAA GAGCAAGCCA AATGTGTGGG 420 CGTACTTCTC TGTCAAGGTAATTTATATCT ATAGNGNCTC TGCTATATAA GTGTTTCACA 480 ATGNTTTAAT TTTNCGGCTACTTTTTTACA GCTGTGGGGC ACCCGNGTCT TGGTTCCATT 540 TGGAAGTNGA TGAAANAATGTTTTA 565 188 amino acids amino acid linear peptide YES N-terminalArabidopsis thaliana pKLU81 14 Leu Ser Gly Lys Ser Leu Val Xaa Ser ThrSer Leu Phe Leu Thr Gly 1 5 10 15 Ser Gly Ser Ser Ser Leu Ser Met HisThr Asn Asn Val Leu Ser Val 20 25 30 Ser Leu Xaa Phe Arg Arg Asp Lys ThrVal Leu Trp Asn Arg Thr Trp 35 40 45 Trp Arg Leu Xaa Phe Leu Ala Lys AsnIle Cys Cys Ser Leu Pro Phe 50 55 60 Xaa Phe Xaa Xaa Ser Ser Ile Tyr LysAla Lys Arg Ser Leu Cys Gly 65 70 75 80 Cys Thr Cys Asp Lys Xaa Gly IleLeu Met Thr Gly Ser Asn Gln Pro 85 90 95 Ala Val Glu Phe Arg Xaa Cys AspIle Cys Gly Met Gln Ser Glu Ser 100 105 110 Ser Phe Thr Glu Glu Leu CysGly Val Thr Leu Val Xaa Thr Cys Asn 115 120 125 Arg Xaa Xaa Val Leu ArgAla Ser Gln Met Cys Gly Arg Thr Ser Leu 130 135 140 Ser Arg Xaa Phe IleSer Ile Xaa Xaa Leu Leu Tyr Lys Cys Phe Thr 145 150 155 160 Met Xaa XaaPhe Xaa Gly Tyr Phe Phe Thr Ala Val Gly His Pro Xaa 165 170 175 Leu GlySer Ile Trp Lys Xaa Met Lys Xaa Cys Phe 180 185 355 base pairs nucleicacid double linear DNA (genomic) NO NO Arabidopsis thaliana 1 FixIIpKLU81 15 TGCCCCCTGG ATGGCATGTG GTGCTTGGAG GGTTGTGGTT GCAAACGTGACAGGCCGTAC 60 ATGCACTGTC CACGTAAGTT CCGCTTACAA AAAATTTGGT TGTACAAGCAATACAGAGAG 120 TAAGAGTACA CATCTCGATG ACTTACCTGC TGTGATTTAA TATTTCAGATATACCGAGAA 180 GTTGAAACTC CTGGAAGAAA CAGTTTAATC TACCACTCAA TAACCAAGAAGGGACCTTTG 240 CATGAAACCC CAATCAGTGA TCAATATAAG CCCCTGGGAT ATCTCGACAGGCAACGTTTA 300 GCAGCAAGGA GGAGTAACAC TACTTATTGC TATGACTTCC CGTTGGTTTGTTACT 355 118 amino acids amino acid linear peptide YES internalArabidopsis thaliana 1 FixII pKLU81 16 Cys Pro Leu Asp Gly Met Trp CysLeu Glu Gly Cys Gly Cys Lys Arg 1 5 10 15 Asp Arg Pro Tyr Met His CysPro Arg Lys Phe Arg Leu Gln Lys Ile 20 25 30 Trp Leu Tyr Lys Gln Tyr ArgGlu Xaa Glu Tyr Thr Ser Arg Xaa Leu 35 40 45 Thr Cys Cys Asp Leu Ile PheGln Ile Tyr Arg Glu Val Glu Thr Pro 50 55 60 Gly Arg Asn Ser Leu Ile TyrHis Ser Ile Thr Lys Lys Gly Pro Leu 65 70 75 80 His Glu Thr Pro Ile SerAsp Gln Tyr Lys Pro Leu Gly Tyr Leu Asp 85 90 95 Arg Gln Arg Leu Ala AlaArg Arg Ser Asn Thr Thr Tyr Cys Tyr Asp 100 105 110 Phe Pro Leu Val CysTyr 115 172 amino acids amino acid linear peptide NO internalArabidopsis thaliana pKLU81 17 Arg Pro Tyr Phe Leu Gln Ala Ala Val ProLeu Leu Tyr Pro Cys Thr 1 5 10 15 Arg Ile Met Tyr Cys Leu Phe Leu PheAsn Phe Val Glu Ile Arg Arg 20 25 30 Phe Tyr Gly Ile Glu His Gly Gly GlyTyr Asp Ser Trp Arg Lys Thr 35 40 45 Ser Val Val Ala Phe Pro Phe Asp PheAsp Lys Ala Gln Ser Ile Arg 50 55 60 Pro Lys Gly His Cys Val Ala Val ArgVal Thr Ser Glu Xaa Pro Asp 65 70 75 80 Asp Gly Phe Lys Pro Thr Ser GlyArg Val Gln Val Met Glx Tyr Leu 85 90 95 Trp Asn Ala Lys Glx Lys Phe IleHis Glx Gly Thr Leu Trp Gly Asn 100 105 110 Thr Cys Met Asn Leu Gln GlnXaa Leu Ser Phe Lys Ser Lys Pro Asn 115 120 125 Val Trp Ala Tyr Phe SerVal Lys Val Ile Tyr Ile Tyr Xaa Xaa Ser 130 135 140 Ala Ile Glx Val PheHis Asn Xaa Leu Ile Xaa Arg Leu Leu Phe Tyr 145 150 155 160 Ser Cys GlyAla Pro Xaa Ser Trp Phe His Leu Glu 165 170 169 amino acids amino acidlinear peptide NO internal Rattus rattus 18 Pro Arg Leu Gln Val Glu HisPro Cys Thr Glu Met Val Ala Asp Val 1 5 10 15 Asn Leu Pro Ala Ala GlnLeu Gln Ile Ala Met Gly Ile Pro Leu Phe 20 25 30 Arg Ile Lys Asp Ile ArgMet Met Tyr Gly Val Ser Pro Trp Gly Asp 35 40 45 Ala Pro Ile Asp Phe GluAsn Ser Ala His Val Pro Cys Pro Arg Gly 50 55 60 His Val Ile Ala Ala ArgIle Thr Ser Glu Asn Pro Asp Glu Gly Phe 65 70 75 80 Lys Pro Ser Ser GlyThr Val Gln Glu Leu Asn Phe Arg Ser Asn Lys 85 90 95 Asn Val Trp Gly TyrPhe Ser Val Ala Ala Ala Gly Gly Leu His Glu 100 105 110 Phe Ala Asp SerGln Phe Gly His Cys Phe Ser Trp Gly Glu Asn Arg 115 120 125 Glu Glu AlaIle Ser Asn Met Val Val Ala Leu Lys Glu Leu Ser Ile 130 135 140 Arg GlyAsp Phe Arg Thr Thr Val Glu Tyr Leu Ile Lys Leu Leu Glu 145 150 155 160Thr Glu Ser Phe Gln Leu Asn Arg Ile 165 120 amino acids amino acidlinear peptide NO internal Gallus domesticus 19 Ala Ala Gln Leu Gln IleAla Met Gly Ile Pro Leu His Arg Ile Lys 1 5 10 15 Asp Ile Arg Val MetTyr Gly Val Ser Pro Trp Gly Asp Gly Ser Ile 20 25 30 Asp Phe Glu Asn SerAla His Val Pro Cys Pro Arg Gly His Val Ile 35 40 45 Ala Ala Arg Ile ThrSer Glu Asn Pro Asp Glu Gly Phe Lys Pro Ser 50 55 60 Ser Gly Thr Val GlnGlu Leu Asn Phe Arg Ser Asn Lys Asn Val Trp 65 70 75 80 Gly Tyr Phe SerVal Ala Ala Ala Gly Gly Leu His Glu Phe Ala Asp 85 90 95 Ser Gln Phe GlyHis Cys Phe Ser Trp Gly Glu Asn Arg Glu Glu Ala 100 105 110 Ile Ser AsnMet Val Val Ala Leu 115 120 75 amino acids amino acid linear peptide YESinternal Arabidopsis thaliana 1 FixII pKLS2 20 Ile Val Asp Ile Ala GluArg Ala Asp Val Asp Ala Val Trp Ala Gly 1 5 10 15 Trp Gly His Ala SerGlu Asn Pro Leu Leu Pro Glu Lys Leu Ser Gln 20 25 30 Ser Lys Arg Lys ValIle Phe Ile Gly Pro Pro Gly Asn Ala Met Arg 35 40 45 Ser Leu Gly Asp LysIle Ser Ser Thr Ile Val Ala Gln Ser Ala Lys 50 55 60 Val Pro Cys Ile ProTrp Ser Gly Thr Gly Val 65 70 75 73 amino acids amino acid linearpeptide NO internal Saccharomyces cerevisiae 21 Cys Val Gln Met Ala GluVal Thr Arg Val Asp Ala Val Trp Pro Gly 1 5 10 15 Trp Gly His Ala SerGlu Asn Pro Glu Leu Pro Asp Ala Leu Asp Ala 20 25 30 Lys Gly Ile Ile CysLeu Gly Pro Pro Ala Ser Ser Met Ala Ala Leu 35 40 45 Gly Asp Lys Ile GlySer Ser Leu Ile Ala Gln Ala Ala Asp Val Pro 50 55 60 Thr Leu Pro Trp SerGly Ser His Val 65 70 32 amino acids amino acid linear peptide YESinternal Arabidopsis thaliana 1 FixII pKLS2 22 Arg Tyr Leu Val Ser AspAsn Ser Asn Ile Asp Ala Asp Thr Pro Tyr 1 5 10 15 Ala Glu Val Glu ValMet Lys Met Cys Met Pro Leu Leu Ser Pro Ala 20 25 30 32 amino acidsamino acid linear peptide NO internal Saccharomyces cerevisiae 23 LysPhe Leu Val Glu Asn Gly Glu His Ile Ile Lys Gly Gln Pro Tyr 1 5 10 15Ala Glu Ile Glu Val Met Lys Met Gln Met Pro Leu Val Ser Gln Glu 20 25 30692 base pairs nucleic acid double linear DNA (genomic) NO NOArabidopsis thaliana lambda FixII pKLS2 24 TCGACTCGAT CTGAAAATATCTAGTGTTCA ACAAACTTCA GATTCTTCGA TCTACATATA 60 AATCTGTTAC ATTCTTTTTTTTATCAAAGA AATCACATTA TTTTAGTAAC TAATCCTAAC 120 TATAAAATCT TTATTCAAGTATTTGATTAT CCTTGATGAA CTTTTAACAA ACGGAATCAA 180 ATATAGGAAA CTAAATCGACCTATACAGAA AAATAATATT TAAATACAAT ACTTTTTTTT 240 CCTACTTAGC ACTTGGATGGCTTTATTGGC TTCATGATCT AGTGGAGCAA GATCAGTAGA 300 GATTTGATAT GGTTCAAGTTTGTTCTGGTC TAGTTTTTAC GGGCATTTTT ATGTACCTCG 360 TGAACTTTCA AGTTATAAAATCCCGGTGCC TTGGAAAAAA AAGGTCTCAA AGACATAAGC 420 ATACAATAAA ATTTGTTTTACAAAGTTTGG AACAAGTCAA CGATGATTCG TTAATTTTCA 480 TTGCTAAAAT GATTGGATCATTCACAATTA ACAAAAATGA GGAAAGAATG AGAGAAAGAT 540 GATAAGGTTG CCATACAATATAAACCCATA CCTAACTCTC AACTATATCT CAACCCCCAG 600 TCATTTATAG TTACTATTAAGCCATTAATA TTATTTCTTT GTCAATGAGA CCACTTTTAT 660 TCTCATTTTA AATAATCAAACAAAATGAAG AT 692 395 base pairs nucleic acid double linear DNA(genomic) NO NO Arabidopsis thaliana pKLS2 25 GAACTACTAT TATCTGAATTAACCGTGTTT TACTGTACAG AACACATGTA TTAAGCTCAA 60 TTTCAGCAAT GAAGTTTTGGTCTTTGGAGT TATTTGTCAT TCATCTGAAC ATCTTTGTCT 120 ACAACCTGTG TGCAGATGGCTGAAGTAACA CGCGTGGATG CAGTTTGGCC TGGTTGGGGT 180 CATGCATCTG AAAACCCCGAATTACCTGAT GCCCTAGATG CAAAAGGAAT CATATGTCTT 240 GGTCCTCCAG CATCTTCAATGGCAGCACTG GGAGATAAGA TTGGTTCTTC GTTGATTGCA 300 CAAGCTGCTG ATGTACCCACTCTGCCATGG AGTGGTTCCC ATGTAAGTAA ATTTACTCTT 360 GTTAAGCTTG AGTATTCTATAGTGTCACCT AAATA 395 872 base pairs nucleic acid double linear DNA(genomic) NO NO Arabidopsis thaliana pKLS2 26 GGAGGGTCCA ATTACTGTGCTCCGCCAGAA ACTTTTCAAG AAACTTGAAC AAGCAGCTAG 60 AAGGTTGGCT AAGAGTGTTAACTATGTTGG AGCTGCTACT GTTGAGTATC TCCACAGTAT 120 GGACACTGGG GAGTACTACTTCTTAGAGCT TAACCCTCGC TTACAGGGTG GTTTCATACT 180 GCAGCTTTTT GCGTTGAAATATAATGAAGG TCCGGACTTG AAAATTGAAT GACTTGTTTA 240 ACTTGATGTT TGAGGTCAGGTTGAGCATCC TGTCACTGAG TGGATTGCCG AGATAAATCT 300 TCCTTCTGCC CAAGATATACTGTGGGGATG GGAATTCCTC TCTGGCAAAT CCCTGGTATA 360 ATCTACGTCC TTATTTCTTACAGGCAGCGG TTCCTCTTCT TTATCCATGC ACACGAATAA 420 TGTACTGTCT GTTTCTCTTTAATTTCGTAG AGATAAGACG GTTCTATGGA ATAGAACATG 480 GTGGAGGTTA TGATTCTTGGCGAAAAACAT CTGTTGTAGC CTTCCCTTTT GATTTTGATA 540 AAGCTCAATC TATAAGGCCAAAAGGTCATT GTGTGGCTGT ACGTGTGACA AGTGAGGATC 600 CTGATGACGG GTTCAAACCAACCAGCGGTA GAGTTCAGGT AATGTGATAT CTGTGGAATG 660 CAAAGTGAAA GTTCATTCACTGAGAACTCT GTGGGTAACA CTTGTATGAA CTTGCAACAG 720 GAGTTGAGTT TTAAGAGCAAGCCAAATGTG TGGGCGTACT TCTCTGTCAA GGTAATTATA 780 TCTATAGAGA CTCTGCTATATAAGTGTTTC ACAATGTTTT AAATTTTACG ACTACTTTTT 840 TACAGTCTGG TGGAGGCATCCACGAGTTCT CG 872 1641 base pairs nucleic acid double linear DNA(genomic) NO NO Arabidopsis thaliana pKLS2 27 CTATGTAAGA ACCTCTTTCTCAGAGATTTA TTTGTCTTGA AAAGTTTCTA TCTGGTGACG 60 AAATGTTCTA TCTGTCCAGAAAGCATCAGC GACCAGTGCT GCTGTGGTTT CAGATTACGT 120 TGGTTATCTG GAGAAGGGGCAAATCCCTCC AAAGGTAATC CAATACCAGG GATCTCTTTT 180 GCCTTTCTAG TGATGTTCTTGTAGCTAACT TTTTCTCTCT TAACTTGCAG CATATATCTC 240 TTGTACATTC TCAAGTGTCTCTGAATATTG AAGGAAGTAA ATATACGGTA TTCGCCTACT 300 ATCCAAATTT TACGTCTCTGCAATTTCGTA TTTTCCTCTG CCATATTATT TTTGCGCTGA 360 AGATATTGTT ACCAGGCTTACTAACATGAA CATAACTGTT CTAGAGTGAT TAGCAATGTA 420 GTCCGGGGTG GATCAGGAACCTACAGGCTA AGAATGAACA AGTCAGAAGT GGTAGCAGAA 480 ATACACACTC TACGTGATGGAGGTCTGTTG ATGCAGGCAA GTTTTCTGCC TTTTTCTATA 540 CTACAAGACA AGGACATACATGTGTCGCGC AGAAAAAAAC TTCTGGAGAA TCTCACTTCC 600 TTTTCTTGTT TTCACTGTCATTGCAGTTGG ATGGCAAAAG CCATGTGATA TATGCAGAGG 660 AAGAAGCTGC AGGAACTCGTCTTCTCATTG ATGGAAGAAC TTGTTTGCTA CAGGTTTCTG 720 CTAATTTTTT TGTGTGTTTACCATTTTACT TCACGTTTCT CTGAAGTCAT CTTTAGCTTT 780 TAAGCTGTCT GTCAATTTTGGCTTATTCAG AATGACCATG ATCCATCAAA GTTAATGGCT 840 GAGACACCGT GCAAGTTGATGAGGTATTTG GTTTCTGACA ACAGCAATAT TGACGCTGAT 900 ACGCCTTATG CCGAAGTTGAGGTCATGAAG ATGTGCATGC CACTTCTTTC ACCTGCTTCA 960 GGAGTTATCC ATCTTAAAATGTCTGAAGGA CAAGACATGC AGGTTCACTT CATTGCTAAA 1020 CAAAAAGTCT ACAGTTCTGTTTAAATTGAT TAACCCATCC ATTATTTTTT TCACAGGCTG 1080 GTGAACTTAT CGCCAATCTTGATCTTGATG ATCCTTCTGC TGTAAGAAAG GCCGAACCCT 1140 TCCATGGAAG TTTCCCAAGATTAGGGCTTC CAACTGCAAT ATCCGGTAGA GTTCATCAGA 1200 GATGTGCCGC AACATTAAATGCTGCACGCA TGATTCTTGC TGGCTATGAG CATAAAGTAG 1260 ATGAGGTAAA CACTGTTTGTTTTTCCTATT TGATCCAACT CTCTCTACTA GATTATTTGA 1320 CTATGAGATA GCTCATACGTCGCAGGTTGT TCAAAGACTT ACTTAATTGC CTTGATAGCC 1380 CTGAACTCCC ATTTCTTGCAGTGGCAACAG TGCTTTGCAG TTCTGGCGAC ACGACTACCT 1440 AAAAATCTCA GGAACATGGTAAACACCTGT GTAGTATTCA TAATCCGGTT CTTATATATT 1500 GATATTTGTT TTGAGTTCAAGACTTTTAAT CATATCTAAA TAAAACTCTT TATCAGCTAG 1560 AATCAAAGTA TAGGGAATTTGAGAGTATTT CCAGAAACTC TTTGACCACC GATTTCCCTG 1620 CCAAACTTTT AAAAGGCAGT C1641 725 base pairs nucleic acid double linear DNA (genomic) NO NOArabidopsis thaliana pKLS2 28 CGAGTCAATT ACTTGAACAG ACCAAACTAAGTGAAGCTTC GTTCAAACAT TGCTAGAAGC 60 CTTTCAGAGT TAGAAATGTT TACAGAGGACGGAGAAAATA TGGATACTCC CAAGAGGAAA 120 AGTGCCATTA ATGAAAGAAT AGAAGATCTTGTAAGCGCAT CTTTAGCTGT TGAAGACGCT 180 CTCGTGGGAC TATTTGACCA TAGCGATCACACACTTCAAA GACGGGTTGT TGAGACTTAT 240 ATTCGCAGAT TATACCAGGT TCGAGTTCATTCTTCCGCAC CCTTATTGTT CAAAATTCTT 300 TTTGTACTGC AATTGATTAC AGAAAATTTTGACTTCATTT TAACCCGACT CTTGTCATCA 360 GCCCTACGTC GTTAAAGATA GCGTGAGGATGCAGTCGCGC CGGATGCAGT GGCACCTTTC 420 TGGTCTTCTT GATTCCTGGG ATTTCCTAGAGGAGCATATG GAAAGAAAAA ACATTGGTTT 480 AGACGATCAC GACACATCTG AAAAAGGATTGGTTGAGAAG CGTAGTAAGA GAAAATGGGG 540 GGCTATGGTT ATAATCAAAT CTTTGGAGTTTCTTCCACGT ATAATACGTG CAGCATTGAG 600 AGAAACATAG CACAACGACT ATGAAACTGCCGGAGCTCCT TTATCTGGCA ATATGATGCA 660 CATTGCTATT GTCGGGCATC AACAACCAGATGAGTCTGCT TCAGGACAGG TACTTGACAC 720 AGTAT 725 830 base pairs nucleicacid double linear DNA (genomic) NO NO Arabidopsis thaliana pKLS2 -Region E 29 ACCGAGAAGT GAACCTGAAG AAACAGTTTA ATCTACCACT CAATAACCCAAGAAGGGACC 60 TTTGCATGAA ACCCCAATCA GTGATCAATA TAAGCCCCTG GGATATCTCGACAGGCAACG 120 TTTAGCAGCA AGGAGGAGTA ACACTACTTA TTGCTATGAC TTCCCGTTGGTTTGTTACTG 180 AATTCATAAG ATTCACACAT ACGCTTACTC TTTTGGCTAT TTCCAACCCCCCTTATGTTA 240 TTTCTTTCCT TTTCAGGCAT TTGGGACAGC CTTGGAACTG TTGTGGGCATCACAACACCC 300 AGGAGTTAAG AAACCATATA AGGATACTCT GATCAATGTT AAAGAGCTTGTATTCTCAAA 360 ACCAGAAGGT TCTTCGGGTA CATCTCTAGA TCTGGTTGAA AGACCACCCGGTCTCAACGA 420 CTTTGGAATG GTTGCCTGGT GCCTAGATAT GTCGACCCCA GAGTTTCCTATGGGGCGGAA 480 ACTTCTCGTG ATTGCGAATG ATGTCACCTT CAAAGCTGGT TCTTTTGGTCCTAGAGAGGA 540 CGCGTTTTTC CTTGCTGTTA CTGAACTCGC TTGTGCCAAG AAGCTTCCCTTGATTTACTT 600 GGCAGCAAAT TCTGGTGCCC GACTTGGGGT TGCTGAAGAA GTCAAAGCCTGCTTCAAAGT 660 TGGATGGTCG GATGAAATTT CCCCTGAGAA TGGTTTTTCA GTATATATACCTAAGCCCTG 720 AAGACCACGA AAGGATTGGT CATCTGTCAT TTGCCCATGA AGGTAAAGCTCCCTAGTGGG 780 GGAAACTAGG GTGGGGTGAA TTGATACGGT CGTTGGGCAA AGAAGGATGG830 764 base pairs nucleic acid double linear DNA (genomic) NO NOArabidopsis thaliana pKLS2 - Region F 30 GCAAGCTCGA AATTAACCCTCACTAAAGGG AACAAAAGCT GGAGCTCTCT TGTAAAAAAC 60 GTAAGAGAAG CATCTGGAGACAACTTAGCA TATAAATCTT CAATGCGTCT GATTCAGGAT 120 TGGTTCTGCA ACTCTGATATTGCAAAGGGG AAAGAAGAAG CTTGGACAGA CGACCAAGTG 180 TTCTTTACAT GGAAGGACAATGTTAGTAAC TACGAGTTGA AGCTGAGCGA GTTGAGAGCG 240 CAGAAACTAC TGAACCAACTTGCAGAGATT GGGAATTCCT CAGATTTGCA AGCTCTGCCA 300 CAAGGACTTG CTAATCTTCTAAACAGGGTA TAAAACAAAA CCCCCCAAAA AAACAAGGTT 360 TTGGTCCCCA AGTAATCCTAACCTGTATGC CGGTTTTTAA AGCCCTAAGT AAATATTTGT 420 GATGCAGGTG GACCGTCGAAAAAGAGAAGA GCTGGTGGCT GCTATTCGAA AGGTCTTGGG 480 TTGACTGATA TCGAAGACTTTAGCTTCTAA TCCAAGAAAG ATGGACATTT AAAGTTTGCT 540 TGTGTCCATT TGGACCATCTTCCTTATATT TGTTGGTCAC AGTTGTAAAT GTTGTTGTAG 600 CTTTGTCATT TCCGTATAAACAAATTACGC AATAATTCAT TCAACATGTC ACTCTTGCTT 660 CATATTTATA CACTGAACCAAGACAATATA ATAGTCTAAA TATAAAACTG ATCGGTCGAC 720 GCCCTATAGT GAGTCGTATTAAGCCGGCCG CGAGCTCTAG AGTC 764 796 amino acids amino acid single linearpeptide NO NO internal Brassica napus Embryo Binding-site 388..392 31Ala Gly Arg Arg Leu Ala Lys Ser Val Asn Tyr Val Gly Ala Ala Thr 1 5 1015 Val Glu Tyr Leu Tyr Ser Met Asp Thr Gly Glu Tyr Tyr Phe Leu Glu 20 2530 Leu Asn Pro Arg Leu Gln Val Glu His Pro Val Thr Glu Trp Ile Ala 35 4045 Glu Ile Asn Leu Pro Ala Ala Gln Val Ala Val Gly Met Gly Ile Pro 50 5560 Leu Trp Gln Ile Pro Glu Ile Arg Arg Phe Tyr Gly Ile Glu His Gly 65 7075 80 Gly Gly Tyr Asp Ser Trp Arg Lys Thr Ser Val Leu Ala Ser Pro Phe 8590 95 Asp Phe Asp Lys Ala Glu Ser Ile Arg Pro Lys Gly His Cys Val Ala100 105 110 Val Arg Val Thr Ser Glu Asp Pro Asp Asp Gly Phe Lys Pro ThrSer 115 120 125 Gly Lys Val Gln Glu Leu Ser Phe Lys Ser Lys Pro Asn ValTrp Ala 130 135 140 Tyr Phe Ser Val Lys Ser Gly Gly Gly Ile His Glu PheSer Asp Ser 145 150 155 160 Gln Phe Gly His Val Phe Ala Phe Gly Glu SerArg Ala Leu Ala Ile 165 170 175 Ala Asn Met Val Leu Gly Leu Lys Lys AsnGln Asn Arg Gly Lys Ile 180 185 190 Arg Thr Asn Val Asp Tyr Thr Ile AspLeu Leu His Ala Ser Asp Tyr 195 200 205 Arg Glu Asn Gln Ile His Thr GlyTrp Leu Asp Ser Arg Ile Ala Met 210 215 220 Arg Val Arg Ala Glu Arg ProPro Trp Tyr Leu Ser Val Val Gly Gly 225 230 235 240 Ala Leu Tyr Lys AlaSer Ala Thr Ser Ala Ala Val Val Ser Asp Tyr 245 250 255 Val Gly Tyr LeuGlu Lys Gly Gln Ile Pro Pro Lys His Ile Ser Leu 260 265 270 Val His SerGln Val Ser Leu Asn Ile Glu Gly Ser Lys Tyr Thr Ile 275 280 285 Asp ValVal Arg Gly Gly Ser Gly Ser Tyr Arg Leu Arg Met Asn Asn 290 295 300 SerGlu Val Val Ala Glu Ile His Thr Leu Arg Asp Gly Gly Leu Leu 305 310 315320 Met Gln Leu Asp Gly Lys Ser His Val Ile Tyr Ala Glu Glu Glu Ala 325330 335 Ala Gly Thr Arg Leu Leu Ile Asp Gly Arg Thr Cys Leu Leu Gln Asn340 345 350 Asp His Asp Pro Ser Lys Leu Met Ala Glu Thr Pro Cys Lys LeuLeu 355 360 365 Arg Tyr Leu Val Ser Asp Asn Ser Ser Ile Asp Ala Asp MetPro Tyr 370 375 380 Ala Glu Val Glu Val Met Lys Met Cys Met Pro Leu LeuSer Pro Ala 385 390 395 400 Ser Gly Val Ile His Phe Lys Met Ser Glu GlyGln Ala Met Gln Ala 405 410 415 Gly Glu Leu Ile Ala Lys Leu Asp Leu AspAsp Pro Ser Ala Val Arg 420 425 430 Lys Ala Glu Pro Phe His Gly Gly PhePro Arg Leu Gly Leu Pro Thr 435 440 445 Ala Ile Ser Gly Lys Val His GlnArg Cys Ala Ala Thr Leu Asn Ala 450 455 460 Ala Arg Met Val Leu Ala GlyTyr Glu His Lys Val Asp Glu Val Val 465 470 475 480 Gln Asp Leu Leu AsnCys Leu Asp Ser Pro Glu Leu Pro Phe Leu Gln 485 490 495 Trp Gln Glu CysPhe Ala Val Leu Ala Thr Arg Leu Pro Lys Asp Leu 500 505 510 Arg Met MetLeu Glu Ser Lys Tyr Arg Glu Phe Glu Ser Ile Ser Arg 515 520 525 Asn SerLeu Thr Ala Asp Phe Pro Ala Lys Leu Leu Lys Gly Ile Leu 530 535 540 GluAla His Leu Leu Ser Cys Asp Glu Lys Asp Arg Gly Ala Leu Glu 545 550 555560 Arg Leu Ile Glu Pro Leu Met Ser Leu Ala Lys Ser Tyr Glu Gly Gly 565570 575 Arg Glu Ser His Ala Arg Val Ile Val His Ser Leu Phe Glu Glu Tyr580 585 590 Leu Ser Val Glu Glu Leu Phe Asn Asp Asn Met Leu Ala Asp ValIle 595 600 605 Glu Arg Met Arg Gln Gln Tyr Lys Lys Asp Leu Leu Lys IleVal Asp 610 615 620 Ile Val Leu Ser His Gln Gly Ile Lys Asp Lys Asn LysLeu Val Leu 625 630 635 640 Arg Leu Met Glu Gln Leu Val Tyr Pro Asn ProAla Ala Tyr Arg Asp 645 650 655 Lys Leu Ile Arg Phe Ser Thr Leu Asn HisThr Asn Tyr Ser Glu Leu 660 665 670 Ala Leu Lys Ala Ser Gln Leu Leu GluGln Thr Lys Leu Ser Glu Leu 675 680 685 Pro Ala Ser Asn Ile Ala Arg SerLeu Ser Glu Leu Glu Met Phe Thr 690 695 700 Glu Asp Gly Glu Asn Met AspThr Pro Lys Arg Lys Ser Ala Ile Asn 705 710 715 720 Glu Arg Met Glu AspLeu Val Ser Ala Ser Leu Ala Val Glu Asp Ala 725 730 735 Leu Val Gly LeuPhe Asp His Ser Asp His Thr Leu Gln Arg Arg Val 740 745 750 Val Glu ThrTyr Ile Arg Arg Leu Tyr Gln Pro Tyr Val Val Lys Glu 755 760 765 Ser IleArg Met Gln Trp His Arg Ser Gly Leu Ile Ala Ser Trp Glu 770 775 780 PheLeu Glu Glu His Ile Phe Arg Lys His Trp Leu 785 790 795 2391 base pairsnucleic acid double linear cDNA to mRNA NO NO Brassica napus EmbryopRS6, pRS8 32 TGGCTGGTAG AAGGTTGGCT AAGAGTGTTA ACTATGTTGG AGCAGCTACTGTTGAATATC 60 TCTACAGCAT GGACACGGGG GAGTACTACT TCTTAGAGCT TAACCCTCGGTTACAGGTTG 120 AGCACCCTGT AACTGAATGG ATTGCCGAGA TAAATCTTCC TGCTGCGCAAGTTGCTGTTG 180 GGATGGGAAT TCCTCTCTGG CAAATCCCTG AGATAAGACG GTTCTATGGTATAGAACATG 240 GTGGAGGTTA CGATTCTTGG AGGAAAACAT CTGTGCTAGC CTCCCCTTTTGATTTTGATA 300 AAGCTGAATC TATAAGGCCA AAAGGTCATT GTGTGGCTGT ACGCGTGACAAGTGAGGACC 360 CTGATGACGG ATTCAAACCC ACCAGCGGTA AAGTACAGGA GTTGAGTTTTAAAAGCAAGC 420 CAAATGTGTG GGCTTACTTC TCTGTCAAGT CTGGTGGAGG CATCCACGAGTTCTCAGATT 480 CCCAATTTGG CCATGTTTTT GCATTTGGGG AATCCAGAGC CTTGGCAATAGCAAATATGG 540 TCCTTGGGCT TAAAAAAAAT CAAAATCGTG GAAAAATTAG GACTAACGTTGACTACACGA 600 TTGACCTTTT ACATGCTTCT GATTACCGGG AAAACCAAAT TCACACTGGTTGGTTGGACA 660 GTAGAATTGC TATGCGGGTC AGGGCAGAGA GGCCTCCATG GTACCTCTCTGTTGTCGGAG 720 GGGCTCTCTA TAAAGCATCA GCGACCAGTG CTGCTGTAGT CTCGGATTATGTTGGTTATC 780 TAGAGAAGGG ACAAATTCCC CCAAAGCATA TATCTCTTGT GCATTCTCAAGTGTCTCTGA 840 ACATTGAAGG AAGTAAATAT ACGATTGATG TGGTCCGGGG TGGATCAGGAAGCTACAGGC 900 TAAGAATGAA CAACTCAGAA GTTGTAGCAG AAATACACAC TCTACGTGATGGAGGTCTGT 960 TGATGCAGTT GGATGGTAAA AGCCATGTGA TATATGCAGA GGAAGAAGCTGCAGGAACCC 1020 GTCTTCTTAT TGACGGAAGA ACTTGTTTAC TTCAGAATGA TCACGATCCTTCAAAGTTGA 1080 TGGCTGAGAC ACCGTGCAAG CTGCTGAGGT ATTTGGTTTC AGATAATAGCAGTATTGATG 1140 CTGACATGCC CTACGCGGAA GTTGAGGTCA TGAAGATGTG CATGCCACTTCTTTCACCTG 1200 CATCAGGAGT TATACATTTC AAAATGTCTG AAGGACAAGC CATGCAGGCTGGTGAACTTA 1260 TAGCCAAGCT TGATCTTGAT GATCCTTCTG CTGTAAGAAA GGCCGAACCCTTCCATGGAG 1320 GTTTCCCAAG ATTAGGGCTT CCAACGGCAA TTTCTGGTAA AGTTCATCAGAGATGTGCTG 1380 CAACTTTAAA TGCTGCTCGC ATGGTTCTTG CCGGCTATGA GCATAAAGTAGATGAGGTTG 1440 TTCAAGACTT GCTTAACTGC CTTGATAGCC CTGAACTCCC ATTCCTTCAGTGGCAAGAGT 1500 GCTTCGCAGT TCTGGCAACA CGACTACCGA AAGATCTCAG AATGATGTTAGAATCCAAGT 1560 ATAGGGAATT TGAGAGTATA TCCAGGAACT CTCTCACCGC AGATTTCCCTGCCAAACTTT 1620 TAAAAGGCAT TCTTGAGGCT CATTTATTAT CTTGTGATGA GAAAGATAGGGGTGCCCTTG 1680 AAAGGCTCAT TGAACCATTG ATGAGCCTTG CAAAGTCTTA TGAAGGTGGTAGAGAAAGTC 1740 ATGCCCGTGT TATTGTTCAT TCTCTTTTTG AAGAATACCT ATCTGTAGAAGAATTATTCA 1800 ATGATAACAT GCTGGCTGAT GTTATTGAAC GCATGCGTCA GCAATACAAGAAAGATCTGT 1860 TGAAGATTGT TGATATTGTG CTCTCACACC AGGGCATTAA AGACAAAAACAAACTCGTTC 1920 TTCGGCTCAT GGAGCAGCTT GTTTACCCTA ATCCTGCTGC ATACAGAGATAAACTTATCC 1980 GATTCTCGAC ACTAAACCAT ACTAATTACT CTGAGTTGGC ACTGAAGGCAAGCCAATTAC 2040 TCGAACAGAC CAAATTAAGT GAACTTCCAG CTTCAAACAT TGCTAGAAGCCTGTCAGAGT 2100 TAGAAATGTT TACAGAGGAT GGGGAAAATA TGGATACTCC CAAGAGGAAGAGTGCCATTA 2160 ATGAAAGAAT GGAAGATCTT GTGAGCGCAT CCTTAGCTGT TGAAGATGCTCTCGTGGGAC 2220 TATTTGACCA CAGCGATCAC ACACTTCAAA GACGAGTTGT TGAGACTTATATTCGCAGAT 2280 TATATCAGCC CTACGTCGTC AAAGAAAGCA TCAGGATGCA ATGGCACCGGTCTGGTCTTA 2340 TTGCTTCTTG GGAGTTCCTA GAGGAGCATA TTTTCCGGAA ACATTGGCTT A2391

What is claimed is:
 1. A partial cDNA insert specifying acetyl CoenzymeA carboxylase (ACCase), isolated from seed of Brassica napus, having thenucleotide sequence set forth in FIG. 6 (SEQ ID NO:12) or set forth inFIG. 12 (SEQ ID NO:32) or of the insert contained in the plasmid pRS1,which has been deposited in Escherichia coli under accession no. NCIMB40555, and variations thereof permitted by the degeneracy of the geneticcode which encode the amino acid sequence of the Brassica napus ACCase.2. A partial cDNA specifying acetyl Coenzyme A carboxylase (ACCase),isolated from wheat germ, having the nucleotide sequence set forth inFIG. 4 (SEQ ID NO:9) or of the insert contained in the plasmid pK111,which has been deposited in Escherichia coli under accession no. NCIMB40553, and variants thereof permitted by the degeneracy of the geneticcode which encode the amino acid sequence of the wheat germ ACCase. 3.An isolated genomic DNA specifying acetyl Coenzyme A carboxylase(ACCase) from Arabidopsis thaliana having the nucleotide sequence setforth in FIG. 8 (SEQ ID NO:13) or of the insert contained in the plasmidpKLU81, which has been deposited in Escherichia coli under accession no.NCIMB 40554, and variants thereof permitted by the degeneracy of thegenetic code which encode the amino acid sequence of the Arabidopsisthaliana ACCase.
 4. A gene construct for use in transforming plantscomprising a promoter active in plant cells, a structural regionencoding mRNA in sense or antisense orientation to one or more domainsof the ACCase gene and a 3′ untranslated region, wherein said structuralregion is selected from the group consisting of SEQ ID NO:9, SEQ IDNO:12, SEQ ID NO:13 and SEQ ID NO:32.
 5. A construct as claimed in claim4 in which the promoter is a tissue-specific or developmentallyregulated promoter.
 6. A construct as claimed in claim 4 in which thepromoter is the promoter of the napin gene of Brassica napus.
 7. Amethod of transcribing the structural region of the gene construct asclaimed in claim 4 comprising inducing the promoter of the geneconstruct and thereby transcribing the structural region which is in thesense orientation.
 8. A method of transcribing the structural region ofthe gene construct as claimed in claim 4 comprising inducing thepromoter of the gene construct and thereby transcribing the structuralregion which is in the antisense orientation.
 9. A plant expressioncassette comprising (i) a promoter recognized in a plant and (ii) astructural region encoding one or more domains of a plant acetylCoenzyme A carboxylase (ACCase) enzyme, said structural regioncomprising the isolated partial cDNA of claim 1 or claim 2 or theisolated genomic DNA of claim
 3. 10. A plant expression cassette asclaimed in claim 9 in which the promoter is a tissue-specific ordevelopmentally regulated promoter.
 11. A plant expression cassette asclaimed in claim 10 in which the promoter is a seed-specific promoter.12. A method of transcribing the structural region of the expressioncassette as claimed in claim 9 comprising inducing the promoter of theexpression cassette and thereby transcribing the structural region,which encodes a full-length ACCase enzyme.
 13. A method of transcribingthe structural region of the expression cassette as claimed in claim 12,wherein the structural region is in the sense orientation relative tothe promoter.
 14. A method of transcribing the structural region of theexpression cassette as claimed in claim 12, wherein the structuralregion is in the antisense orientation relative to the promoter.
 15. Amethod of transcribing the structural region of the expression cassetteas claimed in claim 9 comprising inducing the promoter of the expressioncassette and thereby transcribing the structural region, which encodes apartial-length ACCase enzyme.
 16. A method of transcribing thestructural region of the expression cassette as claimed in claim 15,wherein the structural region is in the sense orientation relative tothe promoter.
 17. A method of transcribing the structural region of theexpression cassette as claimed in claim 15, wherein the structuralregion is in the antisense orientation relative to the promoter.